Liquid chromatographic determination of 6-thiopurine metabolites formed in vitro in electrochemical and enzymatic oxidative activation

Abstract

A micellar liquid chromatographic method is described which was developed for the separation of the oxidation metabolites of 6-thiopurine formed in vitro by electrochemical and enzymatic activation. Electrochemical activation was carried out with an electrochemical cell on-line with the chromatograph. In the potential range 0.4–0.8 V vs. Pd, intermediate purine-6-sulfenic acid could be detected together with purine-6-sulfinic acid and 6-thiopurine disulfide. At potentials > 0.8 V, purine-6-sulfonic acid was detected and the oxidation of 6-thiopurine was completed. Intermediates and products formed in the horseradish peroxidase-catalyzed oxidation of 6-thiopurine were also studied. Enzymatic activation with horseradish peroxidase was similar to electrochemical oxidation at <0.8 V. Detection of sulfenic acid in the enzymatic oxidation supports earlier results which indicated that this metabolite may have biological significance. The results also provide some insight into the enzymatic oxidation pathway.