HPLC DETERMINATION OF FIVE CAFFEINE METABOLITES

An HPLC method for the determination of caffeine metabolites in urine was established. Shim Pack CLC-ODS column (5 μm) was eluted with the mobile phase of methanol—acetonitrile—0.05% acetic acid = 12∶1∶87 (v/v) at a flow rate of 1.2 ml·min^-1, and the ultraviolet absorbance was monitored at 280 nm. The 13 caffeine metabolites and caffeine were well separated and the concentrations of the five metabolites, AFMU, 1U, 1X, 17U, and 17X, were determined. The recoveries of the five metabolites were above 87%, the inter- and intra-day variations were less than 3%. The concentrations of the five metabolites in 120 volunteers were determined. The ratios of the metabolites were employed for the assessment of CYP1A2, NAT, and XO enzymes successfully.