Details of a fluorometric method for the separate determination of free and conjugated metanephrine and normetanephrine in urine are presented. The procedure consists of: (1) a separation and concentration of free amines by absorption on a column of Dowex-50 resin followed by elution with a small volume of aqueous ammonia; (2) acid hydrolysis of the initial effluent from the Dowex-50 column to release conjugated amines, which are then separated in the same manner as for free amines; (3) separation of the metanephrine and normetanephrine in each fraction by chromatography on a column of Amberlite CG-50; and (4) a fluorometric estimation of the amines by a variation of the trihydroxyindole method. Metanephrine may be estimated in samples which contain between 0.25 and 50 μg. and normetanephrine between 0.35 and 120 μg. No substances have been encountered in urine which interfere with fluorescence development or which respond to the test. The average daily excretion of the amines by 6 male subjects was: metanephrine, free, 29.5 μg., conjugated, 134.1 μg.; total, 163.6 μg.; and normetanephrine, free, 21.2 μg.; conjugated, 223.2 μg.; and total 244.4 μg. The amounts of the different fractions excreted by an individual appear to be relatively constant, and marked differences in the pattern of excretion was observed in different persons. There were marked diurnal variations in the rate of excretion of the amines but there was no consistent pattern to the variation.
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