Comparative study of HPLC methods for the Analysis of Diterpene Glycosides from Stevia rebaudiana

Extracts of Stevia rebaudiana have demonstrated sweetness up to 300 times greater than table sugar, and have recently been granted approval through the GRAS certification process for use in commercial food and beverage products. With the growing commercial use of Stevia extracts there is an increased need for testing for the known 'impurities' of Stevia extracts. A comparative study is made between the industry accepted JECFA method which utilizes NH₂-columns under isocratic conditions [1] and an improved method, developed by the authors, which utilizes a Phenomenex Synergi Hydro-RP column and a linear gradient [2], for the HPLC analysis of rebaudioside A and related diterpene glycosides found in Stevia rebaudiana.

The improved method offers greater sensitivity and greater resolution of minor constituents and maintains resolution over extended use of the column. The improved method is also readily compatible with additional detection techniques including mass spectrometry (LC/MS) and Evaporative Light Scatting Detection (ELSD), overcoming a limitation of JECFA method. The two methods have been evaluated using standards of rebaudiosides A, B, C, D and F, dulcoside A, isosteviol, isosteviol monoside, steviol, steviol glucuronide, stevioside and steviolbioside.

References: [1] Kolb, N. et al. (2001)J. Agric. Food Chem. 49:4538–4541.

[2] Hoekstra, B., Schaneberg, B. (2007) Graz Poster Presentation.