Objectives: Individual activities of N-acetyltransferase 2 (NAT2) and of xanthine oxidase (XO) can be assessed using ratios of urinary caffeine metabolites. We investigated how ratios changed over time and which urine collection interval would be the best for NAT2 and XO activity assessments. Methods: On two occasions separated by 14days, 16 healthy male Caucasians collected urine before and 0-2, 2-4, 4-6, 6-8, 8-12, 12-16 and 16-24h after a dose of 150mg caffeine given in the framework of a phenotyping cocktail study. The metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U) were quantified with LC-MS/MS. The molar ratio (AFMU + AAMU)/(1X + 1U + AFMU + AAMU) was used as a NAT2 metric, while the ratio 1U/(1X + 1U) served as XO metric. Results: The NAT2 ratios were stable in the intervals 4-24h after caffeine dosing. Mean intra-individual coefficients of variation were 11-23% starting 4h post-dose, while inter-individual variability reached 37-75%. The XO ratios increased gradually by 14% from the 2-4 to the 16-24h interval. The mean intra- and inter-individual coefficients of variation of XO activity were 3-18 and 7-10% respectively. No significant differences between study occasions were observed. Conclusions: Any sampling interval at least 4h after caffeine dosing is suitable for NAT2 and XO activity assessments. XO activities can only be compared between volunteers and studies if the same urine collection schedule has been respected. The low intraindividual variability allows for sample sizes of 16 and 6 participants in crossover interaction studies of NAT2 and XO activity respectivel
