A Modified Spot Test for Urinary Vanilylmandelic Acid by Open-System Paper Chromatography and its Separation Mechanism

概要

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The spot test for urinary 3-methoxy-4-hydroxy-mandelic acid (vanilylmandelic acid: VMA) combined with open-system chromatography was established and its separation mechanism was studied.<BR>Solutions of sample and standard were spotted on a filter paper, Whatman No.1, 15×15cm, and dried. A solution of diazotized 4-nitroaniline in 0.2 N hydrochloric acid was sprayed on the filter paper. The filter paper was placed in a dish containing 50ml of 70% ethanol in such a way that the spot was 1.5cm distant from the surface of the solvent. After development for about 40 minutes, the intensity of the colored spot was compared with of the adjacent standard.<BR>In the present method, the violet colored spot derived from VMA in the sample was separated from the abundunt spray reagent and co-existing uric acid and ascorbic acid.<BR>In this chromatography, the upward decrease of the content of ethanol in the filter paper was clear around the spotted line followed by gradual change. Consequently, the colored VMA spot was separated as a narrow band in the area of steep gradient of the ethanol content.<BR>The present method is selective and convenient enough to be applied to the first screening test for neuroblastoma and pheochromocytoma.