N. Rodopoulos et A. Norman, DETERMINATION OF CAFFEINE AND ITS METABOLITES IN URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS, Scandinavian journal of clinical & laboratory investigation, 54(4), 1994, pp. 305-315
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.