Isolation of 6-mercaptopurine in human plasma by aluminum ion complexation for high-performance liquid chromatographic analysis

A sample preparation technique and a high-performance liquid chromatographic method for 6-mercaptopurine (6-MP) that is simple, sensitive and without interference from its metabolites is described. 6-Thioguanine (6-TG) is added as an internal standard to the plasma sample, which is then treated with an aqueous solution of aluminum perchlorate to denature the plasma proteins and form complexes with 6-TG, 6-MP and its major metabolite, 6-thiouric acid (6-TUA). These complexes coprecipitate with proteins on centrifugation. 6-MP and its analogues are then extracted from the precipitate with perchloric acid containing sodium hydrosulfite and the extract is chromatographed on an Ultrasphere ODS column eluted with 0.1 M phosphoric acid and 0.001 M dithiothreitol in deionized water. The eluate is monitored at 340 nm. No interfering peak was encountered in over 300 clinical plasma samples. 6-TUA was separated from 6-MP and was found to be present in much higher concentration than 6-MP itself throughout the sampling time (6 h) following oral administration of the drug.