Method Development And Validation Of Caffeine And Paraxanthine In Human Plasma

By Rachel Sun, Orlando J. Bravo, and Brian J. Engel

• Caffeine and paraxanthine quantitation method is validated
• The interference of theobromine and theophylline is evaluated
• It is necessary to separate paraxanthine from theophylline
• The method has been applied to several clinical studies successfully

Caffeine and its metabolites are ubiquitous in human biological fluids due to dietary consumption. The metabolites of caffeine are paraxanthine, theobromine and theophylline. The ratio of paraxanthine to caffeine may serve as a surrogate measurement of liver function, and thus, there is much interest in monitoring these two analytes simultaneously. Often times, the interference of theophylline to paraxanthine is overlooked for many LC/MS/MS methods although theophylline and paraxanthine have the same MS/MS transition. Thus, chromatographic separation of theophylline and paraxanthine is necessary for accurate quantitation of the primary metabolite.