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.     

Liquid chromatographic methods for the quantification of catecholamines and their metabolites in several biological samples—A review

The measurement of catecholamines and their metabolites in biological samples remains a current analytical challenge, in spite of the great diversity of methodologies that have been developed throughout the years. High-performance liquid chromatography is the standard method for their separation and quantification in biological samples, either coupled with electrochemical, fluorescence, chemiluminescence or mass spectrometry detection. This review summarizes the most important physicochemical properties of catecholamines, the wide panoply of sample preparation techniques and the main issues to consider during the development of chromatographic methods. The major difficulties encountered during the optimization of these procedures are related with the high tendency of catecholamines to oxidize and the very low quantities at which they exist in biological matrices. Herein, the most important aspects that ought to be considered during collection, treatment and storage of fluid and tissue samples intended for catecholamine analysis are underlined, the chromatographic conditions are compared and the technical advantages and limitations of each detection system are discussed.     

Improvements in automated analysis of catecholamine and related metabolites in biological samples by column-switching high-performance liquid chromatography

Previously two fully automated methods based on column switching and high-performance liquid chromatography have been described, one for plasma and urinary catecholamines and the other for catecholamine urinary metabolites. Improvements in these methods, after 3 years of routine application, are now reported. The sample processing scheme was changed in order to eliminate memory effects and, in the procedure for plasma catecholamines, a pre-analytical deproteinization step was added which enhances the analytical column lifetime. The applied voltages for the electrochemical detector have been optimized, resulting in an automated method, suitable for the simultaneous determination of vanillylmandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid. The sensitivity of the methods allows the detection of 2-3 ng/l of plasma catecholamines and 0.01-0.06 mg/l of urinary metabolites. Also, it is possible to switch from one method to the other in only 30 min. The normal values obtained from 200 healthy people are reported, together with a list of 57 potential interfering substances tested.     

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.     

Chromatographic methods for the analysis of basic neurotransmitters and their acidic metabolites.

Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC-MS and the reversed-phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC-MS is the method of choice. In addition the limits of detection of GC-MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC-MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.     

On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis

Analysis of low concentrations of metabolites is required for new fields of biological research, such as metabolomics. In this review, recent work in our laboratory aimed at developing improved strategies for on-line sample preconcentration of metabolites by capillary electrophoresis (CE) is presented. Dynamic pH junction, sweeping and dynamic pH junction-sweeping represent three complementary methods for electrokinetic focusing of large volumes of sample directly on-capillary. Focusing selectivity and focusing efficiency are two factors that can be used to assess the suitability of each method for different classes of metabolites. Buffer properties can be selected to enhance the focusing of specific types of metabolites based on knowledge of the analyte physicochemical properties. The application of on-line preconcentration CE for trace analysis of metabolites in real samples of interest, such as biological fluids and cellular extracts, is also demonstrated. Under optimum conditions, up to three orders of magnitude increase in concentration sensitivity can be realized for several classes of metabolites, including catecholamines, purines, nucleosides, nucleotides, amino acids, steroids and coenzymes. Recent work on hyphenating on-line preconcentration with multiplexed CE is highlighted as a promising platform for sensitive and high-throughput analyses of metabolites.     

Application of NMR metabolomics to search for human disease biomarkers

Since antiquity, humans have used body fluids like saliva, urine and sweat for the diagnosis of diseases. The amount, color and smell of body fluids are still used in many traditional medical practices to evaluate an illness and make a diagnosis. The development and application of analytical methods for the detailed analysis of body fluids has led to the discovery of numerous disease biomarkers. Recently, mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and multivariate statistical techniques have been incorporated into a multidisciplinary approach to profile changes in small molecules associated with the onset and progression of human diseases. The goal of these efforts is to identify metabolites that are uniquely correlated with a specific human disease in order to accurately diagnose and treat the malady. In this review we will discuss recent developments in sample preparation, experimental techniques, the identification and quantification of metabolites, and the chemometric tools used to search for biomarkers of human diseases using NMR.     

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 method for comprehensive analysis of urinary acylglycines by using ultra-performance liquid chromatography quadrupole linear ion trap mass spectrometry

Acylglycines are an important class of metabolites that have been used in the diagnosis of several inborn errors of metabolism (IEM). However, current analytical methods detect only a few acylglycines. There is a need to profile these metabolites in a comprehensive manner for studying their functions and improving their diagnostic values for different IEM and potentially other diseases. We describe a sensitive method that combines the chromatographic resolving power of ultra-performance liquid chromatography (UPLC) to separate closely related metabolites including isomers with tandem mass spectrometry (MS/MS). Acylglycines were extracted from urine using an anion exchange solid-phase extraction (SPE) cartridge. After UPLC separation, the acylglycines were detected on a hybrid triple quadrupole linear ion trap mass spectrometer. A set of standards were used for the development of an optimal MS acquisition method. Several acquisition modes using information derived from collisioninduced dissociation breakdown curves were used to detect acylglycines. Using this method, 18 acylglycines were detected in the urine of healthy individuals and confirmed using standards, while 47 additional acylglycines were detected and tentatively identified, based on their retention and fragmentation pattern. Among the 65 acylglycines detected, only 18 of them have been previously reported in biofluids of healthy individuals. These results will be deposited in a public human metabolome database. This example illustrates that by developing a method tailored to the analysis of a class of metabolites sharing similar structural moieties, we can potentially identify many more new metabolites, thereby expanding the overall metabolome coverage.     

人体邻苯二甲酸酯暴露的尿液生物标志物分析方法

邻苯二甲酸酯（phthalates，PAEs）是一类典型的环境内分泌干扰物。近年来，由于PAEs产量和使用量的增加，其对人体健康的危害尤其是生殖发育毒性受到了人们高度关注。由于PAEs在环境、食物（材）中广泛存在，导致人体不可避免地长期暴露于PAEs化合物，因此很有必要开展人体PAEs暴露评估。对人体尿液样品中的PAEs代谢物进行筛选和定量是评价PAEs暴露的重要手段，而建立它们准确、可靠的分析方法是重要前提。目前，邻苯二甲酸单酯和次级代谢物分别是短链和长链PAEs暴露最为常用的生物标志物。离线或在线固相萃取与高效液相色谱-串联质谱联用已成为测定PAEs代谢物的首选方法。本文主要综述了人体PAEs暴露的尿液生物标志物的分析方法，并讨论了这些方法在实际应用中的优点、局限性及挑战。     

Chemical Toolbox to Decode the Microbiota Lexicon

The human microbiota deploys a diverse range of molecules and metabolites to engage in chemical communications with the host, mediating fundamental aspects of host health. Studies of the structures and activities of bioactive molecules produced by the microbiota are imperative to address their implications in microbiota associated diseases in human. By drawing experiences from different research fields, chemists and chemical biologists, who are experts in dealing with chemical molecules, are uniquely positioned to contribute to the emerging knowledge of human microbiota. In this minireview, we discuss the current chemical tools and methods that are pertinent to the discovery of microbiota molecules and metabolites, characterizations of their protein targets, as well as evaluations of their biodistributions in hosts. These are key aspects in understanding the chemical underpinnings of the microbiota‐host interactions that would enable future development of diagnostics and therapeutics targeting the human microbiota.     

Thin-layer chromatographic determination of catecholamines, 5-hydroxytryptamine, and their metabolites in biological samples: a review

A review of methodology for separation, detection, and quantitative determination of catecholamines, 5-hydroxytryptamine, and their acidic metabolites in biological tissue and fluids by thin-layer chromatography is presented. Selected procedures, including fluorometric scanning densitometry for catecholamine acetyl derivatives and color scanning densitometry for acids, are described.     

Pretreatment and one-shot separating analysis of whole catecholamine metabolites in plasma by using LC/MS

Catecholamines are biogenic amines that play an important role in the nervous system. Some catecholamines have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma. The analysis of total catecholamine metabolites should be useful for one-shot screening of multiple aspects of diseases; however, it is difficult to do this, because the catecholamine metabolites are divided into three groups: five amines, one amino acid and three carbonic acids. Catecholamines and small molecules were separated from plasma proteins by an internal-surface reversed-phase column (protein-coated octadeyclsilica column) and were analyzed by liquid chromatography (LC)/mass spectrometry (MS) using electrospray ionization time-of-flight MS. Using a reversed-phase column and hydrophilic mobile phases, we succeeded in the separation of nine catecholamines, all of which had similar structures. These nine substances were eluted in the following order: norepinephrine, epinephrine, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. The reproducibility of this method was acceptable. The highest coefficient of variation was 7.4%. In addition, various types of compounds were separated from and detected in plasma proteins by applying LC/MS. The plasma direct injection method, which uses an internal-surface reversed-phase column and an ion-pair reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that high-performance LC could not succeed in separating and detecting with UV detection. We think that the method can be applied to find new markers in neuroblastoma, by comparing the plasma of patients with that of normal infants. The method can be also used to help in making a diagnosis of other diseases and finding their new makers.     

Routine analysis of catecholamines and metabolites in urine by a liquid chromatographic column switching system

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electrochemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Simulation of chemical metabolism for fate and hazard assessment. I. Approach for simulating metabolism

Information regarding the metabolism of xenobiotic chemicals plays a central role in regulatory risk assessments. In regulatory programmes where metabolism studies are required, the studies of metabolic pathways are often incomplete and the identification of activated metabolites and important degradation products are limited by analytical methods. Because so many more new chemicals are being produced than can be assessed for potential hazards, setting assessment priorities among the thousands of untested chemicals requires methods for predictive hazard identification which can be derived directly from chemical structure and their likely metabolites. In a series of papers we are sharing our experience in the computerized management of metabolic data and the development of simulators of metabolism for predicting the environmental fate and (eco)toxicity of chemicals. The first paper of the series presents a knowledge-based formalism for the computer simulation of non-intermediary metabolism for untested chemicals, with an emphasis on qualitative and quantitative aspects of modelling metabolism.     

Routine Analysis of Catecholamines and Metabolites in Urine by a Liquid Chromatographic Column Switching System

Abstract

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electro-chemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Secondary metabolites isolated from Colletotrichum species

Structural and biosynthetic studies of the metabolites isolated from various Colletotrichum species are reviewed. These fungi are destructive post-harvest pathogens on a wide range of plants including cereals, legumes and fruits. The review includes a detailed study of the biological activity of these metabolites and their role in the development of plant diseases. The literature in this field to the present is reviewed and 60 references are cited.     

Determination of catecholamines and their 3-O-methyl metabolites in mouse plasma

The determination of catecholamines and their 3-O-methyl metabolites in a single mouse plasma is necessary to understand the role of the sympathetic nervous activity, while the inactivation of catecholamines by catechol-O-methyltransferase indicates the activity of blood pressure regulation in animals. Here we report the basal catecholamines and their 3-O-methyl metabolite concentrations obtained from 15 microL of mouse plasma utilizing semi-microcolumn high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence detection system. The concentrations were 6.63 +/- 1.37 pmol/mL plasma, 0.49 +/- 0.10 pmol/mL plasma, 5.25 +/- 2.30 pmol/mL plasma, 3.23 +/- 0.84 pmol/mL plasma, 0.44 +/- 0.11 pmol/mL plasma, and 3.39 +/- 1.67 pmol/mL plasma for norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively (n = 5-7). Further, when blood pressure was reduced by minoxidil, plasma catecholamines were found to be significantly increased by the baroreflex-mediated response in mouse.     

PLS‐DA for compositional data with application to metabolomics

When quantifying information in metabolomics, the results are often expressed as data carrying only relative information. Vectors of these data have positive components, and the only relevant information is contained in the ratios between their parts; such observations are called compositional data. The aim of the paper is to demonstrate how partial least squares discriminant analysis (PLS‐DA)—the most widely used method in chemometrics for multivariate classification—can be applied to compositional data. Theoretical arguments are provided, and data sets from metabolomics are investigated. The data are related to the diagnosis of inherited metabolic disorders (IMDs). The first example analyzes the significance of the corresponding regression parameters (metabolites) using a small data set resulting from targeted metabolomics, where just a subset of potential markers is selected. The second example—the approach of untargeted metabolomics—was used for the analysis detecting almost 500 metabolites. The significance of the metabolites is investigated by applying PLS‐DA, accommodated according to a compositional approach. The significance of important metabolites (markers of diseases) is more clearly visible with the compositional method in both examples. Also, cross‐validation methods lead to better results in case of using the compositional approach. Copyright © 2014 John Wiley & Sons, Ltd.