Quantitative Determination of Steviosides and Sugars in Stevia Supplements using HPLC-UV

Stevia rebaudiana, commonly known as sweetleaf, sugarleaf, or simply stevia, is widely grown for its sweetness. 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. In the United States, S. rebaudiana was banned by the FDA in the early 1990s unless labeled as a supplement, but in 2008 approved rebaudioside A extract as a food additive [1]. S. rebaudiana leaves mainly include stevioside, the content (4–20%) mainly depends on the cultivar and on its growth conditions [2]. Other similar compounds are rebaudioside A, rebaudioside C, dulcoside A, a group of steviol-glycoside type diterpenes. With the aim of quantitative determination of steviosides in S. rebaudiana and its supplements, a HPLC method coupled with a PDA detector is developed for identifying rebaudioside A, stevioside and steviolbioside in S. rebaudiana. The separation was achieved by using C-18 column material with a gradient system composed of water and methanol/acetonitrile, both containing phosphoric 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 Stevia supplements, HPLC-UV method is also used for monitoring the presence of glucose in this study. From Stevia supplements, d-glucose, which should not be present in these types of products, has been detected.

Figure 1: HPLC-UV chromatograms of mixture of three standards (A-1) and Stevia supplements (B-1 to C-1) at UV 205nm [rebaudioside A (1); stevioside (2); steviolbioside (3)]

Figure 2: HPLC-UV chromatograms of mixtures of D/L-glucose derivatives (A-2) and Stevia supplements (B-2 to C-2) at UV 254nm [D-glucose derivatives (4 and 4'); L-glucose derivatives (5 and 5')]

Acknowledgements: This research is supported in part by „Science Based Authentication of Dietary Supplements“ and „Botanical Dietary Supplement Research“ funded by the Food and Drug Administration grant numbers 5U01FD002071–10 and 1U01FD003871–02, and the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58–6408–2-0009.

References: [1] Curry LL, Agency Response Letter GRAS Notice No. GRN 000253 (http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm154989.htm) [2] Geuns JMC, et al. (2000) Recent Res Dev Phytochem 4: 75–88.