Characterisation of Stevia Rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry

Comprehensive two-dimensional liquid chromatography (LC × LC) connected on-line to electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS) was employed for analysis of aqueous extract of Stevia rebaudiana. Different combinations of strong cation-exchange (SCX), amino (NH2), and octadecyl siloxane (C18) stationary phases were tested in the separation of all nine known sweet Stevia glycosides. A combination of C18 as the first-dimension column and NH2 as the second-dimension column fully separated all the glycosides from the matrix. The method proved to be quantitative and repeatable. The limit of detection (S/N = 3) for stevioside, a widely used natural sweetener, was 43.4 ng/g in dry leaves. The RSD for retention times was <0.1% and that of peak areas 4.5%.