Urinary 3-methoxy-4-hydroxymandelic acid as measured by liquid chromatography, with on-line post-column reaction

We describe a method for measurement of 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid, VMA) in urine. After the pH of the urine is adjusted to 2.7 and the sample is filtered, exactly 15 microL is injected onto a C-18 reversed-phase column. VMA is eluted from the column with 10 mmol/L phosphate buffer, pH 2.7, containing 30 mL of acetonitrile per liter. The eluate stream is combined with alkaline periodate and then passed through a 60 degrees C water bath. The VMA is completely oxidized to vanillin, which is detected and quantitiated by its absorbance at 360 nm. No deterioration of the column was noted after 167 such injections of urine samples. Long-term control data indicate a CV of 12 and 10% at VMA concentrations of 1.5 and 5.8 mg/L, respectively. Although results correlate well (r = 0.976) with those by the method of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)], they average 10% lower. Of 20 compounds tested, only methyl dopa interfered with the procedure as described.