DETERMINATION OF CAFFEINE AND ITS METABOLITES IN URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS

Caffeine (CA) and its 14 main metabolites were determined in urine by reversed-phase high-performance liquid chromatography (RP-HPLC) and ca pillary electrophoresis (CE). After addition of 1,3,9-trimethylxanthin e, uracil and beta-hydroxyethyltheophylline as internal standards, sam ples were separated by RP-HPLC into three fractions; A, B and C. The f ractions were concentrated by lyophilization and analysed quantitative ly by CE. Fraction A contained 5-acetylamino-6-amino-3-methyluracil, 3 -methyluric acid, 7-methyluric acid and 1-methyluric acid. Fraction B contained 7-methylxanthine, 3-methylxanthine, 3,7-dimethyluric acid, 1 -methylxanthine, 1,3-dimethyluric acid and 3,7-dimethylxanthine, while fraction C contained I,7-dimethyluric acid, 1,7-dimethylxanthine, 1,3 -dimethylxanthine, 1,3,7-trimethyluric acid and caffeine itself. The d etection limit for the various metabolites ranged from 2-5 mu moll(-1) . The within-run precision for the metabolites ranged between 3.6% and 15.2%. The combination of HPLC and CE techniques was found to be a pr actical and specific method for determination of CA and its metabolite s in urine.