Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography

Journal of Chromatography. B, Biomedical Sciences and Applications
C Krul, G Hageman

Abstract

An isocratic high-performance liquid chromatography procedure was developed for the analysis of five urinary metabolites of caffeine; caffeine or 1,3,7-trimethylxanthine (137X), paraxanthine or 1,7-dimethylxanthine (17X), 1,7-dimethylurate (17U), 1-methylxanthine (1X), 1-methylurate (1U) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU). A standardized procedure was used for oral intake of caffeine and for urine collection. Conditions for sample storage and preparation were optimized, resulting in no detectable loss of caffeine metabolites after storage of the urine samples for four months. Urine samples were extracted with chloroform-2-propanol (4:1, v/v) and separated on a reversed-phase column with acetic acid (33%)-tetrahydrofuran-acetonitrile-water (1:2.5:44:925.5, v/v) as the eluent. Peaks were monitored at 280 nm. Peak heights were measured and the five metabolites were quantified using calibration curves. Cytochrome P4501A2 (CYP1A2) activity was calculated from the molar ratio (AFMU+1X+1U)/17U, N-acetyltransferase (NAT) from the ratio AFMU/1X, XO from the ratio 1U/1X+1U and cytochrome P4502A6 (CYP2A6) from the ratio 17U/(17U+17X+1U+ 1X+AFMU). The inter-assay coefficients of variation ranged from 1.7% for 17U to 5.7%...Continue Reading

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