Study on determination of caffeine and theobromine in beverages, beverage ingredients, and pharmaceuticals by high-performance liquid chromatography with diode array detection (HPLC-DAD)

A simple and reliable analytical method for the determination of caffeine and theobromine in beverages (e.g., cola drinks, bottled tea drinks, energy drinks), beverage ingredients (e.g., cocoa powder, green coffee beans, roasted coffee, matcha powder, bagged tea), and pharmaceuticals (e.g., analgesic and antipyretic drugs) was developed and validated by using high-performance liquid chromatography with diode array detection (HPLC-DAD). The cola and energy drinks were degassed, diluted, and filtered before analysis, while a cleanup step utilizing magnesium oxide (MgO) was applied to the bottled tea drinks. The beverage ingredient samples were extracted with hot water and incorporated with MgO clean-up. The pharmaceuticals were ultrasonicated with a mixture of water/methanol (70:30, v/v). Caffeine and theobromine were completely separated on a C18 column (15 cm × 4.6 mm × 5 μm) with isocratic elution using mobile phase as water/methanol (70:30, v/v) mixture at a flow rate of 0.7 mL/min. Analytical signals of the two analytes were recorded at a wavelength of 272 nm. The instrumental method exhibited good precision (RSD < 5%), linearity (R2 > 0.999 over concentration range of 5–500 mg/L), and adequate detection limits (LOD = 1 mg/L). The entire analytical methods for different sample matrices showed acceptable recovery (89–106%) and repeatability (RSD < 5%). The validated methods were applied to analyze caffeine and theobromine in 40 real samples. In most drink and drug samples, measured concentrations of caffeine were in good agreement with label claims (bias < 10%).