Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry.

摘要: Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction xenobiotics. Ratios different caffeine metabolites urine or other body fluids are frequently used characterise the individual/actual activity enzymes. common analytical method involves extensive sample preparation, followed HPLC–UV. presence numerous UV-absorbing chemicals affects sensitivity selectivity this method. We have developed an HPLC–electrospray-MS–MS for determination 11 two internal standards after simple, extractionless preparation. Blank urine, obtained 5 days on methylxanthine-free diet, contained small amounts some metabolites, but no components producing any confounding signals. Eleven were recovered at 90 110% addition blank (0.1 2.5 μM final involving 20-fold dilution urine) 0.1–2.5 concentration range. Other 5-acetylamino-6-amino-3-methyluracil (AAMU) 5-acetylamino-6-formylamino-3-methyluracil (AFMU), detected with similar recovery precision, required higher concentrations (3 30 μM). AFMU was completely converted into AAMU short alkalisation urine. explored six healthy individuals consuming coffee (4 mg per kg mass). These experiments demonstrated simplicity, high under conditions phenotyping.