Simultaneous determination of azathioprine and 6-mercaptopurine in serum by reversed-phase high-performance liquid...

The simultaneous determination of azathioprine and its metabolite 6-mercaptopurine in serum by reversed-phase high-performance liquid chromatography is described. 6-Mercaptopurine was converted to a derivative, 6-mercaptopurine-N-ethylmaleimide, which is stable against autoxidation, on reaction with N-ethylmaleimide. Since the N-ethylmaleimide derivative was more hydrophobic than the parent compound, it could be extracted into ethyl acetate together with azathioprine and the derivative was retained on the reversed-phase column better than 6-mercaptopurine. In addition, 6-mercaptopurine-N-ethylmaleimide absorbed at the same wavelength (280 nm) as azathioprine. Consequently, this derivatization procedure enabled the simultaneous extraction, separation, and detection of these compounds.