Simultaneous assay of the methylxanthine metabolites of caffeine in plasma by high performance liquid chromatography.

A fast and sensitive method by high performance liquid chromatography for the quantification of the methylxanthine metabolites of caffeine in human plasma is described. Plasma is extracted with 20% isopropanol in chloroform and the evaporated residue is redissolved and chromatographed on silica gel using dichloromethane containing 2.2% of a solution of methanol-ammonium formateformic acid (100:0.02:0.017). The method is suitable for metabolic and pharmacokinetic studies of all mono- and dimethylxanthines in plasma. The limits of quantification, with C.V. of 10% or less are 40 ng/ml for the dimethylxanthines, 100 ng/ml for 3- and 7-methylxanthine and 500 ng/ml for 1-methylxanthine.