Samples were taken every ten minutes for two hours, and in a second experiment every hour for 24 hours, and the concentration of caffeine was analyzed via HPLC (Agilent Technologies, 1260 Infinity Series). The concentration of caffeine was calculated directly comparing the area of the peaks measured via HPLC with the calibration curve obtained with caffeine standards of known concentration. The integration of the peaks was performed manually. A C18 column was used (Poroshell 120 EC-C18, 2.7 mm, 4.6 x 50 mm), and isocratic elution was chosen. The mobile phase was 75:25 acetonitrile: deionized water, and a UV-visible detector was employed (DAD Detector Signal (mAU) = 273/4 nm, Reference 360/80 nm). A calibration using caffeine solutions of known concentration was performed. The retention time of caffeine in the column used for this study was approximately 0.54 minutes.