Determination of steviol glycosides in commercial extracts of Stevia rebaudiana and sweeteners by ultra-high performance liquid chromatography Orbitrap mass spectrometry.

Stevia rebaudiana extracts are used as sweeteners in several countries worldwide. Several extracts of diverse composition are available on the market, and their taste depends on the contents of the various steviol glycosides. This study presents an accurate method for the qualitative and quantitative determination of steviol glycosides in 40 Stevia extracts, 7 sweeteners and 3 Stevia-sweetened beverages by a UHPLC coupled to an Orbitrap mass spectrometer. The sub-2 μm amide column provided the separation of all the target analytes in a run time of 30 min with high resolution. The effect of different eluent compositions on the ionisation efficiency of the steviol glycosides was studied. The optimal ionisation conditions were achieved in negative mode using 0.05% formic acid. Under this condition, adducts were not found, [M-H] were the main ions and the spontaneous loss of a glucose residue at C19 was reduced. The %RSD for intra- and inter-day precision for all eleven analytes varied from 2.1 to 4.2% and 3.0-5.1%, respectively. The recoveries from spiked Stevia extract samples were greater than 95% for all analytes. Rebaudioside A was the most abundant, ranging from 23 to 102%. Nine Stevia extracts and one drink were not compliant with the European Regulation. Isosteviol was under the LOD in all samples and steviol was found in four samples in quantities in the range 0.01-0.03%.