Quantitative Determination of Glucose and Five Steviol Glycosides from Stevia Supplements

Steviol glycosides are naturally occurring sweeteners in the extracts of Stevia rebaudiana Bertoni, a plant native to Paraguay and commonly known as sweetleaf, sugarleaf, or simply stevia. As a natural sweetener and sugar substitute, S. rebaudiana extracts have up to 300 times the sweetness of sugar, and garnered attention with the rise in demand for low-carbohydrate, low-sugar food alternatives. Stevia has been used in Japan, China, Korea and Brazil for years as a food additive or sweetener [1]. Steviol glycosides under certain conditions are considered generally recognized as safe by the FDA and have been approved in the European Union. S. rebaudiana leaves mainly include a group of steviol-type diterpene glycosides such as stevioside, rebaudioside A and dulcoside A, etc [2].

With the aim of quantitative determination of steviol glycosides in S. rebaudiana and its supplements, an UPLC-UV-ELSD/MS method was developed for identifying rebaudioside A, stevioside, rebaudioside D, dulcoside A and steviolbioside in S. rebaudiana. The separation was achieved by using Waters high strength silica type column material with a gradient system composed of water and acetonitrile, both containing acetic acid. The column temperature was maintained at 40 °C. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). This method is successfully applied for the analysis of Stevia dietary supplements. Meanwhile, for the purpose of quality assurance of glucose level in Stevia supplements, UPLC-UV-MS method is also used for quantitative determination of glucose from Stevia supplements. Out of 35 tested samples, glucose was found from six products in the range of 0.9 – 91.5% (w/w).

Fig. 1: UPLC-ELSD chromatograms [rebaudioside A (1); rebaudioside A (2); stevioside (3); dulcoside A (4) and steviolbioside (5)]

Fig. 2: UPLC-UV chromatograms of glucose and products derivatives at UV 243nm [Glucose Derivatives (GD) and Internal Standard (IS; fucose derivatives)]

Acknowledgements: This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 5U01FD004246, the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612. References: [1] Kolb N, Herrera JL, et al. (2001) J Agric Food Chem, 49: 4538 – 4541. [2] Wolwer-Rieck U, Tomberg W, et al. (2010) J. Agric Food Chem, 58: 12216 – 12220.