Characterization of diaminouracil metabolites of caffeine in human urine.

A major caffeine metabolite (A1) has been isolated from human urine by a combination of solvent extraction, reverse phase high-pressure liquid chromatography, and silica gel open column chromatography. The chromatographic and spectral properties of A1 are identical with an authentic sample of 5-acetylamino-6-amino-3-methyluracil. A stable isotope-labeling study, in which [2-14C]caffeine in a 1:1 mixture of unlabeled caffeine and [1,3-15N-8-13C] caffeine was administered to subjects, demonstrated that the C-8 carbon of caffeine was lost during the biotransformation to A1. Thus, the acetyl group in A1 does not originate from caffeine. The 1:1 ratio of the mass spectral ions for A1 isolated from the stable isotope study and the specific activity of A1 support caffeine as the principal source of A1. A related metabolite (A2) was too labile for complete purification and structural identification. The chromatographic and mass spectral properties of the degradation product of A2 were identical with those of A1.