Optimization of Micelle-mediated extraction and cloud point pre-concentration for the simultaneous determination of caffeine, theophylline and theobromine in Green tea leaves by High Performance Thin Layer Chromatography

Abstract

In this paper, a micelle-mediated extraction and cloud point preconcentration method was developed for the determination of the purine alkaloids caffeine, theophylline and theobromine in unfermented Camellia sinensis leaves (Green tea) by high performance thin liquid chromatography. The non-ionic surfactants Genapol X-080 and Triton X-114 were tested as extraction solvents and no back-extraction or liquid chromatographic steps were used to remove the targeted purine alkaloids from the surfactant-rich extractant phase. A uniform experimental design approach was demonstrated using the Boxe-Behnken design for the optimization of experimental factors involved in the micelle-mediated extraction process. The separation of the purine alkaloids was achieved on silica gel 60F 254 HPTLC plates using methylene chloride-ethyl acetate-methanol-ammonia (10:10:1:0.05, v/v/v/v) as the mobile phase. The quantitation of caffeine, theophylline and theobromine was carried out using the densitometric reflection/absorption mode at 275 nm. The method was validated for peak purity, precision, accuracy, and robustness. The proposed technique is a low-cost, simple, and sensitive method with high cleanup effect. Finally, the method was successfully applied to separate and determine purine alkaloids in Green tea extracts even at very low concentration levels compared to conventional solvents like methanol.