Simultaneous Determination of Urinary Vanillylmandelic Acid and 5-Hydroxyindoleacetic Acid

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Abstract
A method for simultaneous quantitative determination of vanillylmandelic acid (VMA) and 5-hydroxyindoleacetic acid (5-HIAA) in urine by means of high-voltage paper electrophoresis was described. The extraction with ethyl acetate, coloration with diazotized p-nitroaniline, elution and spectrophotometric determination was carried out in the same way as used for VMA. VMA was separated at first from other urinary compounds including 5-HIAA by electrophoresis using pH 3.6 buffer, then isolation of 5- HIAA was carried out chromatographically on the same filter paper to the right-angle direction. For this purpose, filter paper which was cut into a crosswise form was employed. This method is highly specific and less time consuming, so that it may be useful in studies on catecholamine and serotonin metabolism. The recovery rate was about 86% for 5-HIAA and 94% for VMA, and the error of measurement was about 5% respectively by this method. Estimation revealed that the excretion of 5-HIAA in human urine per day ranged from 1.9 to 5.1 mg with an average of 3.04[plus or minus]1.07 mg in 20 normal subjects, and that the urinary excretion ratio of 5-HTAA/VMA was about 11.22.