High-performance liquid chromatographic determination of dimethylxanthine metabolites of caffeine in human plasma

A normal-phase high-performance liquid chromatographic assay of caffeine and its metabolites, theophylline, theobromine and paraxanthine, in human plasma is described. The two internal standards ethyltheophylline and 1,3,7-trimethyluric acid are used simultaneously and cover the range of different polarities from caffeine to the three dimethylxanthines. Plasma (0.5 ml) in the presence of ammonium sulphate is extracted with chloroform--isopropanol (1:1, v/v). The extract is chromatographed with a LiChrosorb Si 60 5-micron column and a mobile phase of dichloromethane containing 2.5% of a formate buffer in methanol. Calibration is performed with six different calibration mixtures which take into account the large plasma concentration differences between caffeine and its metabolites in man. The method is suitable for the simultaneous determination of caffeine and its dimethylxanthine metabolites in plasma of healthy and diseased persons.