High-performance liquid-chromatographic analysis of dopachrome and dihydroxyphenylalanine

A high-performance liquid-chromatographic system for the determination of dopachrome and dihydroxy-phenylalanine (dopa) is described. The retention of dopa and dopachrome on C18 reversed-phase columns was investigated as a function of pH in the mobile phase, and as expected the capacity factors were found to be pH dependent. The chromatographic behavior is explained by the change in net charge and polarity of dopachrome and dopa when pH varies. Satisfactory separation of dopachrome and dopa was obtained. An advantage of the method is that the measurements of dopachrome stability and disappearance are uninfluenced by concomitant formation of melanochromes which, however, is the case when the disappearance is followed by measurement of the decrease in absorbance at 475 nm. The utility of the method is illustrated by following the disappearance of dopachrome as a measure of dopachrome tautomerase activity.