Quantification of steviol glycosides in food products, Stevia leaves and formulations by planar chromatography, including proof of absence for steviol and isosteviol

- HPTLC analysis of steviol glycosides and their breakdown products steviol and isosteviol.
- Assessment of steviol glycosides via HPTLC-ESI-MS.
- Calculated analysis time of 2.6 min and solvent consumption of 0.4 mL per analysis.
- Hyphenation to Aliivibrio fischeri bioassay to obtain information on bioactive compounds.
- Supporting quality control, as marketed samples were falsified with synthetic sweeteners.

Steviol glycosides may degrade in food products under certain processing and storage conditions. Hence, a method was developed that separated in the same chromatographic run seven important steviol glycosides, and additionally as a sum parameter, their reported breakdown products steviol and isosteviol. Through derivatizations with the 2-naphthol and the primuline reagent, the detection was selective and inexpensive. In case needed, the baseline separation of steviol and isosteviol was also demonstrated after a plate cut and subsequent short development (two-step method). The HPTLC method was robust with regard to varying sample matrix loads, as the stationary phase was used only once. A high sample throughput was achieved, i.e. 23 separations were performed in parallel on one plate. The total analysis time took 1 h (30 min application, 15 min separation and 15 min derivatization/densitometry) leading to a calculated analysis time of 2.6 min per sample. The solvent consumption was 8 mL in total (0.4 mL per analysis). HPTLC-ESI-MS was employed for confirmation of the results obtained. Mass spectra were recorded only from the zones of interest, and not from matrix or background, leading to decisive advantages, such as less need for MS cleaning. The optimized HPTLC method was shown to effectively support quality control, as marketed samples may be falsified with cheaper synthetic sweeteners, which was also demonstrated in this study. The accuracy of the densitometric quantification in HPTLC was considered as high, as standards and samples were separated on fresh adsorbent and detected simultaneously under identical conditions, which minimized the influence of errors. Finally, the Aliivibrio fischeri bioassay was employed to obtain information on bioactive compounds in Stevia leaf extracts.