Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC.

6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6-MPNs) are the two major metabolites found in erythrocytes after administration of azathioprine. In an attempt to understand the role of these metabolites in the pharmacologic and toxic activity of thiopurines, we have developed a HPLC method for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes. A simple and rapid treatment procedure based on deproteinization by perchloric acid with dithiothreitol is described. The nucleotides were hydrolyzed to their own bases by heating the sample for 45 min at 100 degrees C. During acid hydrolysis Me6-MP was converted into a compound analyzed on a Purospher RP18-e column with 0.02 mol/L dihydrogenophosphate buffer-methanol as eluents. With this procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN derivatives, respectively, were found.