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Specific ion-exchange chromatography and fluorimetric assay for urinary 3-O-methyldopamine
Abstract:
A technique for the selective extraction of 3-O-methyldopamine, normetanephrine and metanephrine from a single urine sample has been investigated. After hydrolysis of the conjugates, the diluted mixture is passed through a Dowex 50W-X2 column and the methoxylated amines are eluted by means of concentrated ammonia. The eluate, containing metanephrine, normetanephrine and 3-O-methyldopamine is evaporated, and a solution of the residue in borate buffer is fractionated under strictly controlled conditions on an Amberlite CG-50 column. The three amines so separated are estimated by specific fluorimetric methods. The extraction recovery is 80 +/- 3% for pure solutions and 78 +/- 4% for 3-O-methyldopamine added to urine. The fluorimetric procedure, carried out under well-defined conditions, allows the estimation of 10 ng of 3-O-methethyldopamine. The spectral characteristics of the fluorescent derivative are similar to those obtained with dopamine, so that it can be assumed that iodine oxidation of 3-O-methyldopamine demethylates this compound and oxidises the resulting dopamine to the dopamine fluorophore (5,6-dihydroxy-indole). Of the compounds that might interfere in the fluorimetric procedure, dopamine, DOPA and alpha-methyl-DOPA are destroyed by the ammoniacal elution from the Dowex column and 3-O-methyl-DOPA is eliminated in the effluent from the Amberlite column. The elimination of interfering compounds and the improved separation on Amberlite ensure high specificity for this procedure. We have applied the method to normal urine and to pathological urines from patients with adrenergic tumours or untreated and treated parkinsonian subjects; vital information has been obtained on the prognosis of adrenergic tumours. The presence of large amounts of dopamine, normetanephrine and/or metanephrine does not affect the assay for 3-O-methyldopamine. The method is also applicable to rat and dog urine, and can be applied to tissue extracts with little modification.