Determination of caffeine and its metabolites in urine by high-performance liquid chromatography and capillary electrophoresis.

Abstract

Caffeine (CA) and its 14 main metabolites were determined in urine by reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). After addition of 1,3,9-trimethylxanthine, uracil and beta-hydroxyethyltheophylline as internal standards, samples were separated by RP-HPLC into three fractions; A, B and C. The fractions were concentrated by lyophilization and analysed quantitatively 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 1,7-dimethyluric acid, 1,7-dimethylxanthine, 1,3-dimethylxanthine, 1,3,7-trimethyluric acid and caffeine itself. The detection limit for the various metabolites ranged from 2-5 mumol l-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 practical and specific method for determination of CA and its metabolites in urine.