Simultaneous separation of acid metabolites of catecholamines: application to urine and tissue.
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Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.
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The decarboxylated noradrenaline metabolite 3,4-dihydroxymandelic acid [DHMA, 2-(3,4-dihydroxyphenyl)-2-hydroxyacetic acid] occurs in different mammalian tissues, especially in the heart. To elucidate the physiological function of DHMA, the antioxidative and radical scavenging activity was determined by physicochemical and cell-based test systems. In the 2,2-diphenyl-1-picrylhydrazyl assay it shows a 4-fold higher radical scavenging activity compared to the standard antioxidants ascorbic acid, tocopherol, and butylated hydroxytoluene. DHMA is also a very potent superoxide radical scavenger and shows a 5-fold smaller IC(50) value compared to standard ascorbic acid. Again, in most cases the antioxidative power of DHMA against bulk lipid oxidation determined by accelerated autoxidation of oils is much higher than for the standard antioxidants. In soybean oil and squalene a DHMA/alpha-tocopherol mixture (1:1 w/w) shows a synergistic effect. Last but not least, 0.001 and 0.0005% levels of DHMA protect human primary fibroblasts against H(2)O(2)-induced oxidative stress as determined by the 2',7'-dichlorofluorescein assay.
Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.
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Comparative quantitation of plaque organic acids formed in vivo in the course of chewing sucrose and sorbitol-mannitol containing gums disclosed that the hexitol gum did not generate any additional lactic, formic, butyric, propionic, acetic or pyruvic acid beyond baseline (0 time) values. In contrast, sucrose-containing gum produced significant increases in lactic and butyric acids. Quantitatively, acetic acid was the major acid in all plaque samples; the values were comparable for both gums at all times, suggesting it was mainly generated from endogenous sources.
Oxybutynin is rapidly metabolized in rat liver microsomes. Two major primary oxidation products were identified as N-desethyl oxybutynin and oxybutynin N-oxide. Deuterium substituted substrate was used to aid the identification. N-Desethyl oxybutynin was characterized by gas chromatography electron impact mass spectrometry as its trifluoroacetamide derivative and oxybutynin N-oxide was indicated by the presence of a decomposition product, 2-oxo-3-butenyl-2 cyclohexyl-2-phenylglycolate, as elucidated from the gas chromatographic mas spectrometric analysis. The formation of this product from synthetic oxybutynin N-oxide was verified and occurs by two consecutive rearrangements upon thermolysis of the unstable N-oxide. Attempted titanous chloride reduction of oxybutynin N-oxide resulted in the formation of the hydrolytic products 2-cyclohexyl-2-phenylglycolic acid and 4-diethylamino-2-butynol.