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 366nm, see Figure 1, and Stevioside was furthermore identified by TLC-MS as can be seen in Figure2.

Developed and derivatized plate at 366 nm

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

Plate HPTLC Silica gel 60 F254s, 20x10cm
Sample preparation Stevia leaves: extracted in water and filtered with an 0.45μm syringe filter; Sweetener: dissolved in water; cola drink and isotonic drink were decarbonized and filtered with an 0.45 μm syringe filter
Sample application ATS 4 sample applicator (Camag), 6 mm bandwise
Application volume 0.5, 1.0 and 2.0 μL
Mobile phase Ethylacetate / Glacial acid / Formic acid / Water 100/11/11/27
Migration distance 5 cm
Migration time 29 min
Extraction equipment Plate Express (Advion)
Extraction solvent Acetonitrile / Water 95/5 (v/v) + 0.1% Formic acid
Extraction flow 0.2 mL/min
Wavelength UV light at 366 nm
Staining Anisaldehyde-sulfuric acid reagent
MS equipment Single-quadrupole mass spectrometer expression CMS (Advion)
MS detection ESI (+) mode (m/z 50 - 1200)

Chromatographic data

Track Compound Concentration 
Application volume
hRf Detected mass 
1, 2, 3,
11, 12, 13
Stevioside Standard 1.0 0.5, 1.0, 2.0 64 828.5
4, 14 Stevia plant   2   828.5
5, 15 Sweetener   2   828.5
6, 16 cola drink   2   828.5
7, 17 Isotonic drink   2   828.5
8, 9, 10,
18, 19, 20
Rebaudioside A Standard 1.0 0.5, 1.0, 2.0 56  

Ordering Information

Description Product No.
HPTLC Si60 F254s, 20x10 cm 1.15696
Ethylacetate gradient grade for liquid chromatography LiChrosolv® 1.00868
Glacial acid EMSURE® 1.00063
Formic acid LiChrosolv® 1.00264
Water for chromatography (LCMS Grade) LiChrosolv® 1.15333
Acetonitrile hypergrade for LC-MS LiChrosolv® 1.00029
Formic acid for analysis EMSURE 1.00264
Millex®-FH filter, 0.45 μm hydrophobic PTFE, 25 mm, non-sterile SLFH025