A gas chromatographic method for measuring 6-mercaptopurine in serum

A method is presented for the measurement of 6-mercaptopurine (6-MP) in serum. Serum samples containing the drug were treated with dithioerythritol and saturated with ammonium carbonate. 6-MP was then extracted from the serum with a mixture of isopropanol-ethyl acetate (1:1, v/v) containing 0.01% ethanethiol. The solvent was evaporated, the residue dissolved in dilute hydrocholoric acid, washed with chloroform, and extracted with ethyl acetate. 6-MP was derivatized with trimethylanilinium hydroxide and measured gas chromatographically. The sulfhydryl-protecting reagents, dithioerythritol and ethanethiol, were added to prevent the decomposition of 6-MP. The extraction and clean-up method recovered 78 +/- 2% (S.E.) of the 6-MP present. Tracer amounts of [8-14C]6-MP served as the internal standard during the extraction part of the method. Theophyline was used as the internal standard during the gas chromatographic analysis. The standard curve obtained from the gas chromatograph was linear between 0.5 and 20 mug/ml of 6-MP. Serum samples were stored in the freezer for two weeks without significant loss of drug. No interference was encountered from normal serum constituents or xanthines, such as caffeine or theophylline added to serum.