Caffeine is a natural alkaloid recognized as an active central nervous system stimulant. This substance can be found in beverages, medicines, and dietary supplements and its concentration must be controlled to minimize side effects of overdose. High-performance liquid chromatography (HPLC) is a routine method for caffeine determination which employs the octadecylsilyl stationary phase (SiO2-C18) in combination with methanol and acetonitrile solvents. Different from the others, we propose to apply pentafluotophenyl (SiO2-PFP) stationary phase and water-ethanol mobile phase as environmentally benign solvent alternative. Due to the additional interactions with pentafluorophenyl ring of stationary phase, the separation of caffeine from other compounds, such as theophylline and theobromine can be significantly improved. Caffeine retained better by 1.5-2 times as compared to C18- column. Chromatographic column Discovery F5 (4,6 × 250 mm, 5-µm particle size) thermostatted at 40°C and gradient elution from 20 % to 60% ethanol was used for chromatography. The method was validated for two dietary supplements' specificity and showed the linearity in the range of 5-15 mcg/ml, precision of not more than 2,0%, and accuracy of not more than 1,0%.
Key words: HPLC, ethanol, Caffeine, PFP-column, ecofriendly method.
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