Abstract: AIM: To establish a HPLC method with direct injection for determining five major metabolites of caffeine including 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1-methyluric acid (1U), 1, 7-dimethyluricacid (17U) and 1, 7-dimethylxanthine (17X) in the urine and assess the activities of N -acetyltransferase (NAT2), cytochrome P4501A2 (CYP1A2) and xanthine oxidase (XO). METHODS: The contents of five major metabolites of caffeine in the urine were determined by RP-HPLC method. Frequency distribution histogram were drawn by calculating the AFMU /(AFMU +1X +1U), (AFMU +1X +1U) /17U and 1U /(1X +1U) and then evaluated the activity of NAT2, CYP1A2 and XO, respectively. RESULTS: The frequency distribution histograms of NAT2 indicated two distinct distibution, and the subjects were classified as slow acetylators if PAR<0.26 or as fast if PAR >0.26.The frequency distribution of CYP1A2 and XO histograms indicated normal distribution. CONCLUSION: The method is simple, accurate, rapid, and suitable for the determination of metabolites of caffeine in urine. The method can be used to assay the activities of NAT2, CYP1A2 and XO.

Key words: caffeine metabolites, HPLC, N- acetyltransferase, cytochrome P4501A2, xanthine oxidase