High-performance liquid chromatographic method for simultaneous determination of [l-methyl-14C]caffeine and its eight major metabolites in rat urine

A selective and sensitive reversed-phase liquid chromatographic method was developed for the simultaneous analysis of [1-Me-C-14]caffeine and its eight major radiolabelled metabolites in rat urine. The separation of the complex mixture of caffeine metabolites was achieved by gradient elution with a dual solvent system using an endcapped C-18 reversed-phase column, which in contrast to commonly used C-18 reversed-phase columns also allows the separation of the two isomers of 6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil (1,3,7-DAU), a caffeine metabolite of quantitative importance predominantly occurring in rat. As caffeine is metabolised primarily by members of the cytochrome P450 1A (CYP1A) subfamiliy, determination of the pattern of caffeine metabolites in rat urine enables analysis of activities of this important enzyme subfamily in vivo. Since CYP1A is suggested to be involved in the detoxification of bilirubin, the assay may be applied to search for untoxic inducers of CYP1A which might be of pharmacological interest in the treatment of hyperbilirubinaemia. (C) 1999 Elsevier Science B.V. All rights reserved.