Page 54 - Application Handbook - Liquid Chromatography

SCA_280_081

High Performance Liquid Chromatography

UHPLC assay for stevia glycosides

No. SCA_190_015

Gesa J. Schad, Shimadzu Europa GmbH

Introduction Analytical conditions
With today’s growing health awareness, there is an System configuration:
increasing interest in sugar substitutes in various A Shimadzu Nexera X2 UHPLC system was used
areas of the food industry. Stevia, the natural consisting of two quaternary solvent pumps
alternative to artificial sweeteners, extracted from (LC-30AD) with two 5 channel degassers (DGU-
the Stevia rebaudiana plant has just recently been 20A5R), an autosampler (SIL-30AC), and a column
approved for use in the EU. Rebaudioside A is oven (CTO-20AC). The system was also equipped
largely enriched in commercial Stevia products, with an SPD-M30A photo diode array detector.
while natural extract contains Stevioside as the
major component. Hence, an HPLC assay for Method:
quality control purposes, must be able to clearly Column: ACE Excel 2 Super C18, 150 x 2.1 mm
separate this critical peak pair. Mobile Phase: 10 mM NaH2PO4, pH 2.8
A: in H2O and B: in MeCN/H2O
(80:20 v/v)
Gradient: 39.5 – 48 % B: in 4 min
Cycle time: 7 min Flow rate: 0.6 mL/min
Temperature: 50 °C Injection Volume: 1 µL
Sample: 0.04 mg/mL of each compound in
MeCN/H2O (6:94)
Chromatogram
mAU
30 1.837 – Stevioside 2.2885 – Rubusoside Steviolbioside

25 0.800 – Rebaudioside D 1.738 – Rebaudioside A 2.413 – Dulcoside A D 3.302 – Rebaudioside B
20 2.091 – Rebaudioside F 2.247 – Rebaudioside C 3.509 –

15

10

5

0
200nm,4nm
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 min

Conclusion / Result

A robust, selective, and sensitive separation of time of the JECFA approved assay was
nine stevia glycosides in less than a third of the established successfully within two working days.