Effect of exposure to daylight and air on ethyl acetate used in the determination of 4-hydroxy-3-methoxymandelic acid.

The method was that of Pisano et al. (1962) as described by Sandler and Ruthven (1963). 4-Hydroxy-3-methoxymandelic acid was obtained from Calbiochem (A Grade) and ethyl acetate A.R. (Analar) was used. For the purpose of this investigation, an acidified aqueous solution of V.M.A., saturated with sodium chloride, was extracted with ethyl acetate (30 ml.) which had been treated in various ways before use. This was followed by extraction of the V.M.A. from the ethyl acetate into M-potassium carbonate. The extract of V.M.A. was converted to 4-hydroxy-3-methoxybenzaldehyde (vanillin) by heating at 50 C. for 30 minutes together with 0.1 ml. of 2% w/v sodium metaperiodate solution, and, after cooling, 0.1 ml. of 10% w/v sodium metabisulphite solution was added. The volume was made up to 4 ml. with M-potassium carbonate, and the optical density of the vanillin in this solution measured at 360 mjs. The extraction into toluene, which is part of the procedure of Pisano et al. for purifying urinary extracts, was unnecessary in dealing with aqueous solutions and was omitted. As a standard, V.M.A. was added direct to M-potassium carbonate and converted to vanillin without extraction. The optical density readings so obtained were regarded as being 100% values in assessing loss of V.M.A. during the extraction. Samples of ethyl acetate, treated as follows, were examined by the procedure. (1) Taken direct from the supplier's bottle (dark brown