Electrochemical detection catecholamines

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

Multiple electrodes have been used to obtain selectivity in electrochemical detection. An early example involved the separation of catecholamines from human plasma using a Vydac (The Separation Group Hesperia, CA) SCX cation exchange column eluted with phosphate-EDTA.61 A sensor array using metal oxide-modified surfaces was used with flow injection to analyze multicomponent mixtures of amino acids and sugars.62 An example of the selectivity provided by a multi-electrode system is shown in Figure 2.63... 

Raggi MA, Sabbioni C, Casamenti G, Gerra G, Calonghi N, et al. 1999. Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection. J Chrom B 730 201-211. 

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

For a more detailed overview of electrochemical detection, the interested reader is referred to Kissinger [59]. The range of applications snitable for electrochemical detection are discnssed in an excellent review by Pacakova and Stulik [60]. Perhaps the most biomedically important application is the determination of catecholamines in bioflnids [61-63]. Electrochemical detection has also lent itself to the detection of other medically important amines snch as histamine [64] and nenrotrans-mitters [65]. Additionally, electrochemical detection has been applied to the detection of certain antibiotics [66] and environmental carcinogens [67]. 

Assay of catecholamines in urine by ion exchange chromatography with electrochemical detection... 

Catecholamines, nerve transmitters monitored in brain and heart patients, are separated on C18 using octane sulfonate ion pairing in 6% An/water (pH 3) with added EDTA and phosphate. Detection can be at UV, 270 nm, or by electrochemical detection at +0.72 V for maximum sensitivity. Other tyrosine and tryptophan metabolite neurotransmitters such as serotonin, VMA, and HMA can be analyzed with ion pairing and EC detection. 

Jackson et al. [26] set miniaturized, battery-powered, high-voltage power supply conditions in NCE for the separation of dopamine and catecholamines with electrochemical detection. The authors varied platinum working electrode voltage from 25 to 200 V/cm for achieving low limits of detection. 

The suprahypothalamic neurotransmitter level can be assessed by a determination of catecholamines in circumscribed brain areas, the technique requires preparation of frozen tissue and isolation of specific nuclei by the micropunch technique. The catecholamines and indolamines can be measured by a radio-enzymatic methods and by a high-pressure liquid chromatography (HPLC) with electrochemical detection. These mechanistic investigations are mostly initiated due to questions arising from the receptor interaction profile of the drug candidate, they may be required to prove that such receptor interactions truly change the functional state of neurotransmitters (functional expression). Mostly, however, the peripheral effects of such neurotransmitter mechanisms (for instance prolactin secretion) are sufficiently distinct. 

Electrochemical detectors are based upon the volta-metric oxidation or reduction of separated analytes at a micro- or thin-film electrode. A number of pharmacologically active compounds that are aldehydes, ketones, or quinones (such as doxorubicin), or nitro compounds (such as nitrofurantoin) are amenable to reduction at a mercury or platinum electrode electron-rich indole derivatives and catecholamines can be oxidized at these electrodes. An important condition that must be fulfilled for electrochemical detection to be practicable is that the mobile phase must be capable of conducting an electrical current. This makes electrochemical detection particularly useful in reversed-phase liquid chromatography, where buffered water mixed with one or more organic cosolvents is usually the mobile phase. 

Electrochemical detection has matured considerably in recent years and is routinely used by many laboratories, often for a very specific biomedical application. The most popular applications include acetylcholine, serotonin, catecholamines, thiols and disulfides, phenols, aromatic amines, macrocycUc antibiotics, ascorbic acid, nitro compounds, hydroxylamines, and carbohydrates. As the last century concluded, it is fair to say that many applications for which LC-EC would be an obvious choice are now pursued with LC-MS-MS. This only became practical in the 1990s and is clearly a more general method applicable to a wider variety of substances. In a similar fashion, LC-MS-MS has also largely supplanted LC-F for new bioanalytical methods. Nevertheless, there remain a number of key applications for these more traditional detectors known for their selectivity (and therefore excellent detection limits). 

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. 

Electrochemical Detectors. In amperometric electrochemical detectors (see Chapter 4), an electroactive analyte enters the flow cell, where it is either oxidized or reduced at an electrode surface under a constant potential. Electroactive compounds of clinical interest conveniently analyzed by HPLC with electrochemical detection include the urhiary catecholamines (see Chapter 29). In addition, electrochemi-cally active tags (e.g., bromine) are added to compounds such as unsaturated fatty acids or prostaglandins. 

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. ... 

Bouloux PM, Perrett D. Interference of labetalol metabolites in the determination of plasma catecholamines by HPLC with electrochemical detection. Clin Chim Acta 1985 150 111-7. 

Marchese N, Canini S, Fabi L, Famularo L. Paediatric reference values for urinary catecholamine metabolites evaluated by high performance liquid chromatography and electrochemical detection. Eur J Clin Chem Clin Biochem 1997 35 533-7. 

Parker NC, Levtzow CB, Wright PW, Woodard LL, Chapman JF. Uniform chromatographic conditions for quantifying urinary catecholamines, metanephrines, vanillylmandelic add, 5-hydroxyindoleacetic acid, by liquid chromatography, with electrochemical detection. Clin Chem 1986 32 1473-6. 

Pares-Herbute N, Tapia-Arancibia L, Artier H (1989) Ontogeny of the metencephalic, mesencephalic and diencephalic content of catecholamines as measured by high performance liquid chromatography with electrochemical detection. Int J Dev Neurosci 7 73-79... 

Electrochemical detection with ion-pairing adaptations of reversed-phase chromatography are the most common methodologies, and many techniques for measuring urinary catecholamines and metanephrines have been... 

The coupling of ion-exchange chromatography with electrochemical detection has been applied to the measurement of urinary catecholamines. 2,110—113 applications of ion-... 

R.C. Causon and M.E. Carruthers, Measurement of catecholamines in biological fluids by high-performance liquid chromatography a comparison of fluorometric with electrochemical detection, J. Chromatogr, 229, 301-309 (1982). 

H. Weicker, M. Feraudi, H. Hagele and R. Pluto, Electrochemical detection of catecholamines in urine and plasma after separation with HPLC, Clin. Chim. Acta, 141, 17-25 (1984). 

J. Odink, H. Sandman and W.H.P. Schreurs, Determination of free and total catecholamines and salsolinol in urine by ion-pair reversed-phase liquid chromatography with electrochemical detection after a one step sample clean-up, J. Chromatogr, 311, 145-154 (1986). 

C. Sarzanini, E. Mentasti and N. Mario, Determination of catecholamines by ion-pair chromatography and electrochemical detection, J. Chromatogr. A, 671, 259-264 (1994). 

M. Hay and P. Mormede, Determination of catecholamines and methoxycatecholamines excretion patterns in pig and rat by ion-exchange liquid chromatography with electrochemical detection, J. Chromatogr. B, 703, 15-23 (1997). 

E.C.Y. Chan, P.Y. Wee and PC. Ho, Evaluation of degradation of urinary catecholamines and metanephrines and deconjugation of their sulfoconjugates using stability-indicating reversed-phase ion-pair HPLC with electrochemical detection, J. Pharm. Biomed. Anal., 22, 515-526 (2000). 

B.L. Lee, S.K. Chia and C.N. Ong, Measurement of urinary free catecholamines using high-performance liquid chromatography with electrochemical detection, J. Chromatogr., 494,303-309 (1989). 

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