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

Introduction

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.

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

Figure 2. Mass spectrum of stevioside

Conclusion

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.

Chromatography
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
Extraction Equipment	Plate Express (Advion)
Extraction Solvent	Acetonitrile / Water 95/5 (v/v) + 0.1% Formic acid
Extraction Flow	0.2 mL/min
Detection
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

Table 2.

Track	Compound	Concentration  (mg/mL)	Application volume (µL)	hRf	Detected mass  (m/z)
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