Catecholamines assay methods

It appears that the intermediates formed from different catecholamines are of different stability. The intermediate open-chain quinones derived from catecholamines with a primary amino group in the side chain do not appear to undergo intramolecular cyclization very readily and consequently would be able to take part in competing reactions this would account for the fact that in general it is difficult to obtain efficient conversions of such catecholamines (e.g. noradrenaline) into the corresponding aminochromes. This factor is important in catecholamine assay procedures (see Section V, E) and probably explains the wide variability in the apparent efficiency of the noradrenaline oxidation procedures used (as measured by the intensity of the fluorescence of the noradrenolutin obtained by the particular method). The fact that noradrenaline-quinone is relatively more stable than adrenaline-quinone accounts for the formation of entirely different types of fluorescent products from adrenaline and noradrenaline, respectively, in the Weil-Malherbe assay procedure for catecholamines (see Sections IV, H and V, E, 5). 

Reviews of the different assay methods for catecholamines and biogenic amines are not readily available. Gas-chromatographic and spectrofluorometric methods of analyzing catecholamines have been compared. In another comparative study it has been concluded that replacement of personnel employed in t.l.c. 

Catecholamines and Metabolites HPLC, coupled with electrochemical or fiuorometric detection, now provides the most widely used assay method for measurements of urinary or plasma catecholamines in the routine clinical laboratory. Once equipment is pur-... 

Highly sensitive, specific, and reliable assay methods are required for measuring the normally very low concentrations of epinephrine, norepinephrine, and dopamine in plasma. Both unconjugated (free) and sulfoconjugated catecholamines circulate in human plasma and are increased in plasma from patients with pheochromocytomas. Clinical... 

As with the catecholamines, fluorescence methods have also been reported for urinary metanephrine analysis. Fluorescent derivatization of the metanephrines NM and MN by chemical oxidation was based on modification of the trihydroxyindole reaction used for catecholamines. The individual metanephrines were measured following chromatographic separation and fluorescent derivatization or through the formation of differential fluorescent compounds by oxidation at different pH levelsSince the stability of the fluorescent products was variable, with some products decomposing within 10 min,this method has limited application in current practice. Other early methods for analysis of NM and MN included electrophoresis and paper and thin-layer chromatography. These assays were technically complex and had poor analytical sensitivity. 

M. Israel and M. Tomasi, A chemiluminescent catecholamine assay its application for monitoring adrenergic transmitter release, J. Neurosci. Methods., 91, 101-107 (1999). 

Vaarman A, Kask A, Maeorg U. 2002. Novel and sensitive high-performance liquid chromatographic methods based on electrochemical coulometric assay detection for simultaneous determination of catecholamines, kynure-nine and indole derivatives of tryptophan. J Chrom B 769 145-153. 

Colorimetric assays used in endocrinological procedures are also often subject to drug interference. We have observed an interesting interference in a patient with carcinoid. The patient excreted 400 mg of 5-hydroxyindoleacetic acid (5-HIAA) and when a vanillylmandclic acid (VMA) determination was performed by a nonspecific diazo method, the value was reported to be 375 mg. The catecholamines were just above normal. There was an immediate suggestion that the patient also had a pheochromocytoma. However, when a specific chromatographic VMA method was used, the value was found to be within normal limits. Subse-... 

The assay procedure for catecholamines based on the formation of fluorescent products with ethylenediamine, originally described by Weil-Malherbe and Bone, 197, 198, 270 and considered by some workers to be the most sensitive method, suffers to some extent by a lack of specificity since catechol and 3,4-dihydroxymandelic acid give the same fluorophore as noradrenaline. However, most of the interfering compounds can be eliminated by use of suitable extraction procedures. 

Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [84]. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyidopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported [462J. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. 

Indolealkylamines. GC-MS methods applied in studies of the biochemical pharmacology of indoleamines parallel work on the catecholamines. SIM assays for serotonin (5-hydroxytryptamine), 5-methoxytryptamine, JV-acetylserotonin and melatonin (5-methoxy-N-acetyltryptamine) in rat pineal and brain tissue have been described [453,469]. Pentafluoro-propionyl derivatives and structural homologue standardisation were employed with detection limits in the subpicomole range. Estimation of central indoleamine turnover in man currently depends upon metabolite determination in CSF. Ion monitoring determination of indole-3-acetic acid [454] a metabolite of tryptamine, and isotope dilution assays for 5-hydroxyindoleacetic acid (5-HIAA) [455,458] have been reported. Serotonin is converted by central monoamine oxidase to 5-HIAA and the measurement of this metabolite, formerly by fluorimetry, is of interest in patients with CNS disorders [470]. GC-MS has also contributed to the identification of N,N-dimethyltryptamine in vitro [471] and isotope dilution technique has been applied to the measurement of this metabolite in control subjects and in psychiatric patients [472]. 

False neurotransmitters. A number of substances, in some cases structurally related to the biogenic amines, are considered to act as false neurotransmitters and GC-MS methods have been applied to their detection. Thus, p-hydroxynorephedrine, a metabolite of (-l-)-amphetamine, can apparently replace noradrenaline and be taken up and released by a similar mechanism to that involved for the catecholamine. A quantitative SIM assay for this metabolite (as the pentafluoropropionyl derivative) has been used in studies of its formation and localisation in brain regions of the rat [473]. Similar methods have been applied to the detection in neural tissue of the N-dealkylated metabolites of methamphetamine, which depletes brain noradrenaline and fenfluramine which depletes serotonin [474]. 

The determination of catecholamines requires a highly sensitive and selective assay procedure capable of measuring very low levels of catecholamines that may be present. In past years, a number of methods have been reported for measurement of catecholamines in both plasma and body tissues. A few of these papers have reported simultaneous measurement of more than two catecholamine analytes. One of them utilized Used UV for endpoint detection and the samples were chromatographed on a reversed-phase phenyl analytical column. The procedure was slow and cumbersome because ofdue to the use of a complicated liquid-liquid extraction and each chromatographic run lasted more than 25 min with a detection Umit of 5-10 ng on-column. Other sensitive HPLC methods reported in the literature use electrochemical detection with detection limits 12, 6, 12, 18, and 12 pg for noradrenaline, dopamine, serotonin, 5-hydroxyindoleace-tic acid, and homovanillic acid, respectively. The method used very a complicated mobile phase in terms of its composition while whilst the low pH of 3.1 used might jeopardize the chemical stability of the column. Analysis time was approximately 30 min. Recently reported HPLC methods utilize amperometric end-point detection. 

In contrast to the catecholamines, measurements of urinary metanephrines and VMA are still based in some routine laboratories on the early spectrophotometric assays developed by Pisano, Crout, and others in the late 1950s and early 1960s. Despite subsequent development of a variety of preanalytical cleanup and extraction procedures, these assays remain susceptible to analytical interference. They are also restricted to measurements in urine. Another limitation for spectrophotometric or fiuorometric assays of urinary metanephrines is that these methods do not allow separate (fractionated) measurements of normetanephrine and metanephrine. 

Dietary constituents or drugs can either cause direct analytical interference in assays or influence the physiological processes that determine plasma and urinary levels of catecholamines and catecholamine metabolites. In the former circumstances, the interference can be highly variable depending on the particular measurement method. In the latter circumstances, interference is usually of a more general nature and independent of the measurement method (Table 29-6). 

Only a few methods for urinary catecholamines have been published that do not require preextraction prior to analysis." " These methods minimized sample preparation by making use of different precolumn derivatization procedures. The selection of a suitable method for sample preparation prior to analysis by HPLC depends on a number of factors, such as the biological source, the type of column used and the selectivity of the detection method. In cases where the analyte concentration is very low and the analyte is present in a complex matrix (urine or plasma) with interfering compounds, an exhaustive pretreatment may be unavoidable. Sample pretreatment is also essential to ensure the sensitivity and specificity of the assay and protect the analytical column from contamination. 

Because the best reliability is assured by in vivo monitoring, differential pulse voltammetry (DPV) has been applied for in vivo assay of brain catecholamines.168 The method was not very selective since ascorbic acid was the main interfering species. Furthermore, the implantation of an electrode... 

Methods of biological, chemical, and fluorometric assay of catecholamines have been reviewed." Another review dealing with radioimmunoassay of drugs includes amphetamines." Numerous analytical methods and modifications have been applied to various types of phene thy lamine, and these are summarized in Table 2. A convenient derivative of phenylalanine for g.I.c. determination is 2-trifluoromethyl-... 

The detection limit of the autoxidation assay is 0.5 nM superoxide dismutase The oxidation of adrenalin is followed at 480 nm spectrometrically. 850 pi 100 mM carbonate buffer, pH 10 and 100 pi water or sample are mixed. The reaction is started with 50 pi catecholamine stabilized at pH 2. AA/min is followed. Due to its convenience, this method can be employed in homogenates and other biological samples However, the assay can interfere with reduced glutathione, causing an... 

LC with electrochemical detection offers a sensitive method for measuring human plasma catecholamines that is simpler than the existing radioenzymatic assays (Hallman et al, 1978 Fenn et al, 1978 Hjemdahl et al, 1979 Fig. 6). There are also reports discussing the measurement of serum 5-HT (Sasa et al, 1978). 

An Improved Method for the Differential Assay of 3-0-Methylated Catecholamines in Human Urine Using Ion-Pair Extraction and Gas Chromatography Electron Capture Detection Clin. Chim. Acta 92(2) 235-240 (1979) CA 90 182510e... 

The colour reactions resulting from the oxidation of adrenaline and related catecholamines formed the basis of early qualitative and quantitative assay procedures for adrenaline [8-11]. The characteristic yellow-green fluorescence, which develops rapidly when alkaline solutions of adrenaline are allowed to stand in air, was first reported in 1918 [12], and this has become the basis of one of the most widely-used methods for the estimation of catecholamines (i.e. the so-called lutin or trihydroxyindole method... 

Bioassay methods are often both sensitive and specific. Some are indeed so sensitive that they can be used for the assay of extremely small amounts of circulating catecholamines (see Fig. 1. p. 117). For most purposes, however, chemical methods of assay are now widely used. Of the chemical methods available, only fluorescence techniques offer sufficient sensitivity for application to biological materials. For assay, the catecholamines must be converted to suitable fluorescent derivatives, since their native fluorescence is inadequate. There are two chemical procedures in use, both of which have given rise to a host of methods varying only in experimental detail. 

The reactions leading to the formation of highly fluorescent products following the oxidation of catecholamines and condensation with ethylenediamine are very complex, and only the product from NA is shown in Figure 1. This method is more sensitive than the trihydroxyindole method, and the fluorescent products are more stable, but it is a far less specific assay procedure. Ethylenediamine will condense with many catechol compounds to yield fluorescent derivatives. It is, therefore, essential to employ a very rigorous procedure for the isolation of the amines prior to assay. When such a procedure is used, involving, for example, the separation of acetylated catecholamine derivatives by paper chromatography, the ethylenediamine method provides one of the most sensitive assay procedures for DA. 

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