Determination of Stevioside and Rebaudioside A in Stevia Leaves, Beverages and Food Additives Using HPTLC-MS


High Performance Thin Layer Chromatography coupled with Mass Spectrometry (HPTLC–MS) is a very useful analytical technique where samples often can be analyzed with a minimum of sample preparation. This is exemplified here through the determination of stevioside and rebaudioside in beverages and food additives. Rebaudioside A and stevioside are the main ingredients of the Stevia plant extract. Rebaudioside A is the sweetest, least bitter glycoside of Stevia plants. Therefore, in Stevia products, a high proportion of Rebaudioside A is desirable. As of today, a purity of 98% is the highest possible concentration of rebaudioside A in Stevia products.

In this study, different samples have been analyzed; stevia leaves, beverages and an artificial sweetener. The Stevia leaves were extracted in water and filtered through a 0.45 PTFE syringe filter. The cola and the isotonic drinks were first decarbonized by ultrasonication and thereafter filtered. The sweetener was only dissolved in water. After chromatographic separation on the HPTLC plates, the analyte band(s) were eluted using an TLC-MS interface and detected with a mass spectrometer. A second plate was derivatized with anisaldehyde-sulfuric acid reagent.

Results and Discussion

Stevioside (hRf = 64, m/z = 828.5) and Rebaudioside A (hRf = 56) were detected under UV light at 366 nm, Figure 1, and Stevioside was furthermore identified by TLC-MS as can be seen in Figure 2.

Developed and derivatized plate at 366 nm. Chromatographic data shown further down in tabulated form.

Figure 1.Developed and derivatized plate at 366 nm. Chromatographic data shown further down in tabulated form.

Mass spectrum of stevioside

Figure 2.Mass spectrum of stevioside


These results demonstrate that stevioside and rebaudioside A can easily be analyzed by HPTLC-MS with a minimum of sample preparation even in artificial sweeteners, stevia plants, cola and isotonic drinks.

Application Data

Table 1.

Chromatographic data

Table 2.