The Cytochrome P450 CYP1A2 is a central enzyme in the metabolism of drugs and xenobiotics. The overall activity of this enzyme is influenced by a complex array of biochemical, dietary, and genetic factors. One of the simplest ways to probe the overall output of CYP1A2 is to measure the ratio between the concentration of a precursor and a product of its activity. With the growing interest in the Paraxanthine/Caffeine ratio, the need arises to develop improved analytical methods specifically optimized for the rapid and sensitive determination of paraxanthine and caffeine in biological samples. We report a new optimized method for the determination of caffeine and paraxanthine in various human matrices. The method involved direct determination following protein precipitation based on ultra high performance liquid chromatographic separation with tandem mass spectrometric detection (UHPLC-ESIMS/MS). The method offers an improvement in the detection limit over previously published methods by at least 10-fold (0.1 pg), rapid chromatographic separation (ca. 5 min), the utilization of a green chromatographic solvent (5% v/v ethanol), direct determination with little sample preparation, and the employment of isotopically labeled internal standards and qualifier ions to ensure accuracy. Method validation in urine, saliva, and plasma was performed by spiking at various concentration levels where the recovery and repeatability were within ±15% and ±10%, respectively. The method was applied to investigate the levels of caffeine and paraxanthine in volunteers following controlled caffeine administration and to investigate the inter- and intra-individual variability in the paraxanthine/caffeine ratio in volunteers following an unrestricted caffeine diet. In conclusion, the developed UHPLC-ESIMS/MS method is optimized specifically for the simultaneous determination of the paraxanthine/caffeine ratio in multiple biological matrices, offers several advantages over the current methods, and is well suitable for application in large clinical studies.
Keywords: (U)HPLC; Caffeine metabolism; Cytochrome P450; Mass spectrometry.
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