Catecholamines, separation

Fio. 51. Dependence of retention of catecholamines on volume percent acetonitrile in hetaeric chromatography. The ehient is water-acetonitrile at the volume percent indicated containing 0.2% (v/v) sulfuric acid and 0.1% (w/v) sodium dodecyl sulfote. The catecholamines separated are noradrenaline (NA), adrenaline (A). L-3,4-dihydroxyphenylalanine (LD), normetanephrine (NMA), dopamine (DA), metadrenaline (MA), and 3-methoxytyramine (MDA). Column 5- tm octadecyl silica treated with triroethylchlorosilane, 125 X 5 mm i.d. Reprinted with permission from Knox and Jurand (223). ... 

Figure 6.28 Separation of catecholamines. Separator column Zorbax ODS eluent (A)...

Intrinsic UV fluorescence has been used to perform in situ measurements of serotonin. Loss of serotonin from astrocytes can be monitored using wide-field UV fluorescence microscopy after various chemical treatments. In addition, three-photon scanning fluorescence microscopy has been shown to be useful for tracking secretion of serotonin from granules in cultured cells in response to cross-linking of cell surface IgE receptors. Fluorescence from indo-lamines and catecholamines separated with CE has provided a means to measure these species in the low- to mid-attomole range. 

Derivatization of primary and secondary amines using 9-fluorenylmethyl chloroformate to form a nonpolar, uv-absorbing derivative has been reported (90,91). Amphetamine and catecholamine were used as probes to evaluate this procedure. The derivatives were well behaved and allowed separation in a short time. 

Note The dipping solution may also be used as a spray solution [2]. Catecholamines are only separable on silica gel layers as their triacetyl derivatives but they can be separated underivatized on cellulose layers [4]. 

Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

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... 

Simple etching of the capillary end served to decouple the electrophoretic current from that of amperometric detection, permitting quantitation of attomole levels of catecholamines from brain microdialyzates.24 A postcolumn reactor using bromine generated electrochemically in situ has been used in the detection of peptide thiols, such as glutathione and cysteine, separated by capillary electrophoresis.25... 

Pericytes lie periendothelially on the abluminal side of the microvessels (Figure 15.3). A layer of basement membrane separates the pericytes from the endothelial cells and the astrocyte foot processes. Pericytes send out cell processes which penetrate the basement membrane and cover around 20-30% of the micro-vascular circumference [18]. Pericyte cytoplasmic projections encircling the endothelial cells provide both a vasodynamic capacity and structural support to the microvasculature. They bear receptors for vasoactive mediators such as catecholamines, endothelin-1, VIP, vasopressin and angiotensin II. Pericytes become mark-... 

Figure 1-1 shows an example of the detection limits that can be reached with EC detection. The chromatogram shows the separation and detection of the catecholamines noradrenaline (nor), adrenaline (adr), dihydroxybenzylamine (dhba), and dopamine (dopa), using a glassy carbon electrode at a working potential of +0.6 V. The minimum detectable quantity is less than 3 Pg-... 

Tornkvist A, Sjoberg PJ, Markides KE, Bergquist J. 2004. Analysis of catecholamines and related substances using porous graphite carbon as separation media in liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 801 323. 

The list of separations given here cannot be regarded as exhaustive. Many separations done in RPC are reported in such a fashion that it is difficult to discover them as RPC separations using standard methods of searching the literature. Furthermore, the reports of separations of certain chemical classes, e.g. amino acids or catecholamines, are so prevalent that only a fraction of the references were used in the interest of keeping the reference list to manageable size. 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

Another study of the effects of I on the cardiovascular systemic concluded that, in dogs anesthetized with sodium pentobarbital, the response of blood pressure to intravenous administration of I is a resultant of two separate effects a direct myocardial stimulation that was stopped with dichlorolsoproterenol and a stimulation of vascular smooth muscle that results in a slight increase in renal arterial pressure and a slight decrease in renal arterial flow. Neither atropine nor dichlorolsoproterenol affected these vascular effects. Injections of 1 into a jugular vein or a renal artery had no consistent effect on catecholamine concentrations in plasma taken from a femoral artery or a renal vein. In seven experiments in which I at 21-35 mg/kg was injected into a jugular vein, the mean blood pressure increased from 176/125 + 22/11... 

Dopamine metabolism inhibitors interfere with monoamine oxidase and catecholamine-0-methyltransferase. Monoamine oxidase will be discussed separately in chapter 8. 

Since dopamine is present in sympathetic nervous tissue as a precursor of norepinephrine, and it has a separate metabolic pathway that yields homovanillic acid (HVA), tumors such as neuroblastomas may cause elevations of the urinary dopamine and its metabolite HVA. In some cases these elevations have been observed with normal VMA, total catecholamine, and metanephrine. Urinary HVA is usually normal in patients with phenochromocytoma. Increased HVA is found in special fluids of patients with Parkinson s disease treated with L-dopa. 

There are a number of gas chromatographic procedures for the determination of the catecholamines. Methods generally accepted are making the trimethylsilyl ether derivative of the alcoholic group after using diazomethane to prepare the methyl ester and the heptafluorobutyryl derivative. The ethyl esters have been used for the separation of HVA and isoHVA and mass spectrometry applied to study their characterization. 

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