Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.