Assessment of semi-automated catecholamine assay by HPLC: Choice of reverse phase C18 or cation-exchange columns

Summary HPLC with electrochemical detection is useful in the assay of catecholamines in biological samples. Two types of chromatographic column are currently employed by most investigators: reverse phase C-18 (RPC-18) or strong cation-exchange (SCX) columns. Our aim was to evaluate the specificity, sensitivity and reproducibility of plasma and urinary catecholamine assay by these two columns. Our study indicates that both columns give very good performance for clinical purposes but sensitivity, precision at low concentration and specificity seem to be better with ion-exchange columns which are therefore preferred at the present time.     

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%.     

Enzyme based detection systems in column liquid chromatography — Extension of selectivity and sensitivity

Summary Enzyme based detection systems in column liquid chromatography are described using systems based on both enzyme reactors in conjunction with a flow through detection device and enzyme electrodes. Optimizations for making detection principles compatible with chromatographic systems and applications made in our laboratories are presented for the analysis of e.g. carbohydrates, amino acids, ethanol and catecholamines. Gel-permeation chromatography and mass spectrometry are used as complementary techniques for qualitative and quantitative analysis of complex samples.     

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

Summary Dobutamine is one of the synthetic catecholamines acting directly onβ ₁-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.     

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.     

Basal plasma-catecholamine-level determination using HPLC-ED and different sample cleanup techniques

Summary Reversed phase HPLC with electrochemical detection was used for the determination of basal adrenaline, dopamine and noradrenaline levels in human plasma. These compounds demonstrated good stability during different stages of collection and long-term storage. Using a new electrochemical detector and improving mobile phase parameters, we obtained a detection limit of 2 pg per injection. Good separation of dihydroxyphenylacetic acid was also attained, which is important in investigations with intensive care patients. Good accuracy and precision, demonstrated in the daily quality control measurements taken over a five month period, verified the reliability of the chromatographic separation. However, there was a decrease in the recovery of very low amounts of catecholamines, added to fresh frozen plasma that had previously been made catecholamine-free. According to the widely-accepted extraction method of Anton and Sayre, it is argued that the un-known affinity of catecholamines to acid-prepared aluminium oxide (in comparison to catecholamine —protein binding constants) explains the low accuracy in measurement at very low plasma levels. We thus compared this sample preparation method to recently published extraction procedures.     

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.     

Recent advances in methods for the analysis of catecholamines and their metabolites

Catecholamines, for example epinephrine, norepinephrine, and dopamine, are widely distributed and are important neurotransmitters and hormones in mammalian species. Several methods have been developed for analysis of catecholamines and related compounds. Determination of catecholamines in biological fluids has enabled us to clarify the physiological role played by these amines. Catecholamine levels in plasma and/or urine are also useful for diagnosis of several diseases, for example hypertension, pheochromocytoma, and neuroblastoma. This review covers reports from 2000 to the present of methods for the analysis of catecholamines and their metabolites.     

Chromatographic methods for the determination of pesticides in foods.

Chromatography is the most important technique available to the analyst dealing with the determination of pesticide residues in food, feed and environmental samples. Numerous methods for pesticide residues in foods have been developed in the past few years, and this paper reviews some of the most important procedures. A great variety of chromatographic methods, such as solid-phase extractions, column chromatographic clean-up methods, thin-layer, gas, high-performance liquid and supercritical fluid chromatography, and their coupling with sensitive and selective detection methods are surveyed.     

Analysis of organomercury compounds in sediments by capillary GC with atomic fluorescence detection

Analysis of methyl- and ethylmercury (MM and EM) halides in biological and environmental samples is generally performed by gas chromatography with electron capture detection. Tedious sample work-up protocols and poor chromatographic response (using packed columns) have, however, shown the need for the development of new methods in this field. This paper reports a sensitive method, free from these deficiencies, for the determination of methyl- and ethylmercury. The organomercury compounds (MM and EM) are first released from the sample matrix, by the combined action of acidic potassium bromide and cupric ions, and then extracted into dichloromethane. The initial extracts are subjected to thiosulfate clean-up and the organomercury species are isolated as their chloride derivatives by addition of cupric chloride, and subsequent extraction into a small volume of organic solvent. Capillary GC coupled with atomic fluorescence detection provided excellent separation efficiencies for methyl- and ethylmercury and proved to be a very selective and sensitive technique. The absolute detection limit for both MM and EM was found to be 0.2 pg.     

Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic–mass spectrometric methods, but use of the more recent and sensitive liquid chromatography–(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.     

The effect of temperature on the analysis of metanephrine for catechol-O-methyltransferase activity assay by HPLC with electrochemical detection

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.     

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 catecholamines in human serum by micro high-performance liquid chromatography with micro precolumn and dual electrochemical detection

A dual electrochemical detector having two working electrodes (anode and cathode) in parallel--opposed configuration suitable for micro high-performance liquid chromatography was developed for the selective and sensitive detection of catecholamines on the basis of their electrochemical reversibility and catalytic amplification by recycling oxidation and re-reduction. The micro high-performance liquid chromatographic system with a micro alumina precolumn for enriching catecholamines and the dual electrochemical detector in parallel--opposed configuration was successfully utilized for the determination of catecholamines in healthy human serum injected directly after ultrafiltration.     

Evaluation of a sensitive HPLC method for the determination of Malondialdehyde, and application of the method to different biological materials

Summary Reactive oxygen species (ROS), important mediators of cell and tissue injury during inflammation, are produced by several types of inflammatory cells. The formation of ROS can be monitored by detection of lipid peroxidation products. The extremely broad spectrum of biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that enables the sensitive routine quantitation of aldehydes formed in biological materials. Malondialdehyde (MDA) is a by-product of enzymatic eicosanoid formation and an end-product of nonenzymatic peroxidation of polyunsaturated fatty acids with three or more bisallylic double bonds. The determination of the thiobarbituric acid derivative of MDA (TBA-MDA) is a widely used method for estimating overall lipid peroxidation. We describe a rapid, isocratic, simple, and sensitive high-performance liquid chromatographic (HPLC) method with spectrofluorimetric detection for measurement of MDA-TBA in human biological samples such as plasma, urine, wound secretions, amniotic fluid, sputum and tissue samples. By use of this method, picomole quantities of MDA can be readily and specifically detected in different biological materials. Coefficients of variation of repeated MDA-TBA assays were 4.4% within run and 6.9% from run to run. Reference values are given for a variety of human body fluids and for rat tissues.     

An integrated coupled column liquid chromatography system for the determination of leukotriene metabolites in biological samples

Summary A fully integrated chromatographic system was developed for the determination of leukotrienes in biological samples using photodiode-array detection (PDAD), which eliminates time consuming manual sample handling steps. A special solid phase extraction, (SPE) methodology for leucotriene metabolite stability was developed which increased the recoveries and eliminates the contamination risk of biological samples. The inherent instability (autooxidation) of many of the leukotriene mediators, and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By binding the cell supernatants to the functionalities of the SPE support stabilised these mediators. Cell culture samples were eluted through a disposable C₁₈ SPE column. The SPE columns were allowed to thaw and deposited in an automated sample handling unit (ASPEC XL). Desorption of the analytes was followed by a second on-line SPE step, to eliminate remaining interfering matrix compounds. Typical recoveries when stored at −70°C were in-between 55–97% except for (LTE₄) which was found to be around 40% after 72 days of storage. Seven reversed-phase packings were studied. Selectivity factors, as well as the separation efficiencies, were found to differ for the various C₁₈ bonded silica stationary phases. This integrated on-line column liquid chromatographic system was applied to the determination of leukotriene B₄, leukotriene C₄, leukotriene D₄, leukotriene and E₄ in human cell extracts using prostaglandin B₂ as the internal standard. More than 1500 biological samples were analysed. Some validation data are presented for unattended operations.     

Capillary electrophoresis with direct chemiluminescence detection for the analysis of catecholamines in human urine

A rapid and sensitive method for the analysis of three catecholamines by capillary electrophoresis(CE)with directchemiluminescence(CL)detection is described.The detection limits(S/N=3)were 1.3*10-8g/mL for isoprenaline,1.0*10-8g/mL for epinephrine and 2.8*10-8g/mL for dopamine.The proposed method was successfully applied to theanalysis of catecholamines in urine samples of cigarette smokers and nonsmokers.The results showed that there is a close relationbetween the release of dopamine in human body fluids and cigarette smoking/nonsmoking.     

生物样品中儿茶酚胺类物质分析方法的研究进展

儿茶酚胺是人体内重要的神经递质,血浆及尿中儿茶酚胺的浓度水平可用于诊断高血压、嗜铬细胞瘤及成神经细胞瘤等病症,因而准确测定人体生物样品中儿茶酚胺类物质代谢浓度的变化,在疾病的诊断和治疗中具有重要的意义。该文综述了人体液和组织中儿茶酚胺类物质的不同分析方法,包括高效液相色谱法、毛细管电泳法、质谱法、电化学法、化学发光法、荧光光度法等。     

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.     

Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: a decade of research

The proven ability of gel electrophoresis to simultaneously resolve, in a single experiment, many components from complex biological samples, has determined its preference over a variety of well-established chromatographic methods. Therefore, procedures placed at the interface between gel separation and microanalysis have earned increasing significance with respect to the overall success of the microanalytical strategy. The first of these procedures is the detection technique. The most important requirement for compatibility with further analysis or bioapplications is that the staining method does not compromise the chemical integrity and the biological properties of micropurified biomolecules. Procedures for negative detection of proteins with metal salts that have been proven to comply with this condition have been known for about 15 years. Only recently have these procedures been extended to the field of nucleic acids and lipopolysaccharides. The focus of this review is to chronicle the development and current status of the negative or reverse stain procedure based on the in-gel reaction of imidazole with zinc salts and its applications forthe micropurification and analysis of unmodified proteins, nucleic acids and bacterial lipopolysaccharides. We highlight the common aspects in the detection of the three types of biomolecules, and their applications to structural and biological analyses. Emphasis is given on the mechanism underlying imidazole-zinc staining, as it contributes to a deeper understanding of a general detection mechanism with metal salts. Finally, we discuss the latest applications of the techniques in proteomics and their possible impact on the characterization of gel-separated single components from complex lipopolysaccharides.