According to the “Japanese Pharmaceutical Excipients 2018”, quantification of purified stevia extract should be carried out with a column filled with aminopropyl silylated silica gel, while setting the flow rate to make the retention time of stevioside around 10 minutes and meeting following requirements.

The system suitability requirements:
   The elution order to be rubusoside, dulcoside A, stevioside, rebaudioside C, and rebaudioside A 
    Resolution between stevioside and rebaudioside C: ≥ 1.5
    Relative standard deviation (RSD)*: ≤ 1.5 %
    *Injection of 20-µL stevioside standard (n=6)

The HILICpak VG-50 4E confirmed the requirements were met, and thus is suitable for the quantification of purified stevia extract. Compared to regular silica amino columns, VG-40 4E has two major advantages: 1. VG-50 4E reduces the eluent consumption to a half of a regular silica amino column of the same size uses, as it can be operated with less than a half flow-rate of silica amino column. 2. Since VG-50 4E is a polymer-based amino column, it has higher durability than silica-based amino column.


Standard solution
Sample: 20 µL
1. Rubusoside  0.003 %
2. Dulcoside A  0.003 %
3. Stevioside  0.01 %
4. Rebaudioside C  0.003 %
5. Rebaudioside A  0.005 %

Stevioside standard solution
Sample: 20 µL
3. Stevioside  0.05 %

Column       : Shodex HILICpak VG-50 4E (4.6 mm I.D. x 250 mm)
Eluent       : H2O/CH3CN=20/80
Flow rate    : 0.65 mL/min
Detector     : UV (213 nm)
Column temp. : 35 °C