Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometry

Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30-60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes