A comparative study of five different methods for the determination of 3-methoxy-4-hydroxymandelic acid in urine

Five different methods for the determination of 3-methoxy-4-hydroxymandelic acid (vanilmandelic acid, VMA) in urine were compared: a GLC-FID catecholamine metabolite profiling method, an HPLC method with electrochemical detection, the method of Pisano et al. [1962) Clin. Chim. Acta 7, 285-291), a one dimensional paper chromatographic method with diazotized p-nitroaniline staining and the commercially available Bio-Rad VMA by Column Test. The comparison consisted of an imprecision study, a linearity check, a recovery study, a split sample comparison and an interference study. The best results of the imprecision study (n = 8) were found with the Bio-Rad and the HPLC method (within-run imprecision had a coefficient of variation (CV) of 5.1% and 1.4%; between-days CV of 5.9% and 6.0% respectively for values of 32.4 mumol/l and 24.5 mumol/l). The Pisano method had the poorest within-run CV (14.6%) and between-days CV (16.8%) for a value of 23.2 mumol/l. All methods showed good linearity. The mean recovery of the HPLC method was 101.3%; the mean recovery of the other four methods ranged from 93.9%-96.0%. The split sample comparison showed that the accuracy of the HPLC, the GLC and the Pisano method is comparable. The accuracy of the paper chromatographic method and the Bio-Rad method had a positive bias compared with the HPLC method. Especially the positive bias of the Bio-Rad method can be very large. The HPLC method was not influenced by the compounds tested in the interference study, whereas the GLC method in some cases only suffered from overloading problems. The Pisano and the Bio-Rad method were most influenced by the interfering compounds tested. We conclude that the HPLC and the GLC methods are superior to the other three VMA methods. From an analytical point of view HPLC is the method of choice for determining 3-methoxy-4-hydroxymandelic acid in urine.