Catecholamines analytical methods

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. 

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. 

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-... 

It is perhaps a little surprising that improved analytical methods (e.g., high-performance liquid chromatography, proton resonance mass spectrometry) have not led to a reexamination of catecholamines as markers of stress and adrenal medullary function. 

The microdialysis technique requires that a small-diameter (<300 pm) dialysis tube be stereotaxically implanted in a defined brain area. Perfusion of the dialysis tube with Ringer solutions enables diffusion of small molecules down their concentration gradient from the brain extracellular fluid and into the tube. The substances in the collected dialysates can be identified and measured by various analytical methods, either on-line or after storage (4). Extracellular transmitter levels may be measured either under basal conditions or after evoked release using, for example, high KCl concentrations in the perfusate. Because some classes of neurotransmitters, (such as the amino acids and catecholamines) are rapidly cleared from the extracellular space by specific reuptake mechanisms (5), it is often of interest to examine dialysate levels of these transmitters after perfusion with substances that will block these reuptake mechanisms. 

The oxidation reaction has been carried out with (a) iodine at more alkaline pH values than are used in the case of the catecholamines, (b) potassium ferricyanide, but with a higher concentration of zinc ions needed to catalyse the reaction, which works in the case of metanephrine, but not for the nor compound, and (c) periodates. The periodate oxidation gives amino-chromes only at low pH values [131]. Under alkaline conditions, periodate oxidation leads to degradation of the side-chain to give vanillin. This reaction had, in fact, been the basis of earlier analytical methods for (27) and (28) [132, 133]. 

The most commonplace substrates in energy-transfer analytical CL methods are aryl oxalates such as to(2,4,6-trichlorophenyl) oxalate (TCPO) and z s(2,4-dinitrophenyl) oxalate (DNPO), which are oxidized with hydrogen peroxide [7, 8], In this process, which is known as the peroxyoxalate-CL (PO-CL) reaction, the fluorophore analyte is a native or derivatized fluorescent organic substance such as a polynuclear aromatic hydrocarbon, dansylamino acid, carboxylic acid, phenothiazine, or catecholamines, for example. The mechanism of the reaction between aryl oxalates and hydrogen peroxide is believed to generate dioxetane-l,2-dione, which may itself decompose to yield an excited-state species. Its interaction with a suitable fluorophore results in energy transfer to the fluorophore, and the subsequent emission can be exploited to develop analytical CL-based determinations. 

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). 

Electrochemical detection using amperometric or coulo-metric measurement is preferred for specific measurement of small quantities of 5-HIAA a modification of the method developed by Chou and Jaynes is is available on this book s accompanying Evolve site. Like serotonin, the oxidation potential for 5-HIAA is below 0.6 V and must be optimized for each apphcation. Very few interfering compounds are electrochemically active at such low voltage potentials. But if detection of 5-HIAA with other indoles and catecholamines is desired, then hydrodynamic voltammograms for each analyte should be studied to select the minimum potential that achieves maximum specificity. Some HPLC systems use fluorometric detection, with or without derivatization, for a less demanding measurement of 5-HIAA. A method combining fluorometric and electrochemical detection has also been described. ... 

Vaarmann A, Kask A, Maeorg U. Novel and sensitive high-performance liquid chromatographic method based on electrochemical coulometric array detection for simultaneous determination of catecholamines, kynurenine and indole derivatives of tryptophan. J Chromatogr B Analyt Technol Biomed Life Sci 2002 769 145-53. 

Electrophoresis on a macro scale has been applied to a variety of difficult analytical separation problems inorganic anions and cations, amino acids, catecholamines, drugs, vitamins, carbohydrates, peptides, proteins, nucleic acids, nucleotides, polynucleotides, and numerous other species. A particular strength of electrophoi esis is its unique ability to separate charged macromolecules of interest to biochemists, biologists, and clinical chemists. For many years, electrophoresis has been the powerhouse method of separating proteins (enzymes, hormones, antibodies) and nucleic acids (DNA, RNA), for which it offers unparalleled resolution. ... 

Voltammetric methods are of particular interest with regard to the selective determination of analytes in the presence of interferents. As a widely studied case, one can mention the determination of dopamine and other neurotransmitter catecholamines in the presence of interfering compounds, namely, ascorbic acid and/or uric acid. It is known that dopamine exerts a signilicani physiological role as extracellular chemical messenger whose loss in neurons can be associated to serious diseases such as Parkinsonism. Consequently, its determination in vitro and in vivo is an obvious target in neurochemical studies. 

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. 

The value of electrochemical detection following high performance liquid chromatography is compared with that of the more widely used optical methods of detection. Its advantages are illustrated by its application to three areas of clinical chemistry that had previously posed analytical problems. Its sensitivity allowed its use for the measurement of plasma catecholamines. Its selectivity was employed to produce rapid methods for the determination of urinary levels of catecholamine and tryptophan metabolites. Finally, its value for the estimation of urinary oxalic acid is shown. Future developments such as increasing the range of detectable compounds by derivatization are briefly discussed. 

The current method is to be distinguished clearly from the well-known fluorimetric trihydroxyindol assay for catecholamines, which consumes the analyte (i.e., it is a batch analysis) and is not intended to afford real-time response von Euler, U. S. Lishajko, F. Acta Physiol. Scand. 51, 348 (1961). 

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