Greener and Whiter Analytical Procedure for Theobromine and Caffeine Determination in Tea Using Dimethyl Carbonate as an Extraction Solvent and Mobile Phase Constituent in Reversed-Phase Liquid Chromatography.

Abstract

For the first time, dimethyl carbonate was applied as the sole organic solvent, both as an eluent in solid-phase extraction (SPE) and for the simultaneous determination of theobromine and caffeine in tea extracts using reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC). The proposed procedure has been validated and complies with the requirements of green analytical chemistry (GAC). Solid-phase extraction was used to purify the samples, achieving high recovery rates (97-101%) and precision (RSD < 2%). The method showed satisfactory sensitivity (achieving limit of quantitation values of 0.2 ng for theobromine and 0.35 ng for caffeine), although it was poorer compared to the developed conventional HPLC method using methanol, where the values reached 0.045 and 0.05 ng, respectively. The low toxicity and minimal solvent consumption of DMC contributed to a much more beneficial environmental profile of the developed method, as confirmed by AGREEprep, AGREE, and ChlorTox evaluations. Moreover, by switching from conventional HPLC to UHPLC, the remaining principles of GAC were further incorporated by reducing solvent consumption, analysis time, and energy requirements. In addition, the RGB tool highlighted the higher environmental performance of the method using DMC while not compromising analytical quality or economic practicality. The robustness and environmental benefits make this method a viable alternative for the routine analysis of tea ingredients, supporting sustainable analytical practices.