Development and validation of a rapid ultra high pressure liquid chromatographic method for the determination of methylxanthines in herbal infusions

An ultra high pressure liquid chromatographic method coupled with diode array detector (UHPLC–DAD) has been developed and validated for the fast separation and determination of three major methylxanthines, i.e., caffeine, theophylline and theobromine, in various herbal beverages. Isocratic elution using 0.1 vol% formic acid/CH3OH (92.5:7.5, v/v) enabled the completion of the separation cycle in less than 3 min using a flow rate of 0.7 mL/min and a column temperature of 50 °C. Validation of the method included linearity (0.5–50 mg/L), limits of detection (12–35 μg/L) and quantification (40–120 μg/L), precision, matrix effect and accuracy. The percent recoveries ranged between 90 and 108%.