Abstract: AimTo establish a HPLC method for determining five major metabolites of caffeine in the urine, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1-methyluric acid (1U), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X) and assess the activity of cytochrome P-450 CYP2A6. MethodsThe contents of five major metabolites of caffeine in the urine were determined by RP-HPLC method. Frequency distribution histogram was drawn by calculating the 17U/(AFMU+1X+1U+17X+17U) and then evaluated the activity of CYP2A6. ResultsThe frequency distribution histograms of CYP2A6 approximately indicated three distinct groups, the cut of point is 0.23 between fast metabolizer and intermediate type. And the cut of point is 0.15 between slow metabolizer and intermediate type. ConclusionThe method is simple and rapid, suitable for the determination of metabolites of caffeine in urine. The method can be used to assay the activity of CYP2A6.