A method for the estimation of 3-methoxy-4-hydroxymandelic acid (VMA) in urine, based on its oxidation to vanillin, was introduced by Sandler and Ruthven (1, 2) : VMA was isolated from urine by ion exchange and ethyl acetate extraction and converted to vanillin by autoclaving in acid solution, in the presence of aluminum oxide as a catalyst. Vanillin was estimated calorimetrically by its reaction with indole. The method was later modified by Sunderman et al. (3), who used ferricyanide in acid solution at 37"C, and by Pisano et al. (4), who used sodium periodate in alkaline solution at 50Β°C as the oxidizing agent. While the color formation with indole was retained in the method of Sunderman et al. (3)) measurement of the light absorption of vanillin in the region of the 360-380 mp band was recommended by Sandler and Ruthven (2) and by Pisano et al. (4). The specificity of the method was improved by pretreatment of urine with Florisil (3) and by extraction of the vanillin formed with toluene either from acid solution (3) or, better, from neutral solution (4).
In this paper yet another method for the conversion of VMA to vanillin will be described. The oxidizing agent is cupric ion at a pH of about 10. It will be shown that the three methods of oxidation, by ferricyanide, periodate, or copper, give essentially identical results which compare well with the results obtained by the isotope dilution method of Weise et al. ( 5). It is confirmed that the absorption at 360 rnp is probably more specific than the color reaction with indole. While it is not claimed that the copper method offers any significant advantage over the other two methods, it presents some interesting features which arc thought to be worth recording. EXPERIMENTAL A&oxidation of VMA in the Presence of Catalytic Amounts of Cupric Ion and Other Heavy Metals The present investigation arose from a study of the conversion of 3,4-485