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The stability of the two steviol glycosides stevioside and rebaudioside A and the possible formation of the aglycon steviol in different soft drinks were analyzed in samples spiked with stevioside or rebaudioside A after 24, 48, and 72 h storage times at 80 °C. Degradation of up to 70% was observed, and stevioside was less stable than rebaudioside A. Stevioside and rebaudioside A and their degradation products were analyzed by high-performance liquid chromatography with ultraviolet detection (UV-HPLC) on a HILIC analytical column, and the identity of the degradation products was confirmed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS(n)) in negative mode. A UV-HPLC method was developed using a C18 analytical column to exclude the presence of the aglycon steviol, which gave a positive response in the forward mutation assay using the sensitive Salmonella typhimurium TM677 strain. The recoveries of steviol with this method ranged from 95.9 to 109.2%, and the calibration curves were linear from 1 to 100 μg/mL with R(2) = 0.9999. The limit of detection was 1 μg/mL. Confirmation by LC-ESI-MS(n) resulted in a LOD of 6 ng/mL. The absence of steviol in the degraded samples could be unambiguously confirmed by UV-HPLC and by LC-ESI-MS(n).

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