An improved HPLC method for the analysis of diterpenoid glycosides in Stevia rebaudiana

Stevia rebaudiana, also known as sweetleaf, has been used as a sweetner for centuries in South America. Extracts are up to 300 times sweeter than table sugar, which is attributed to the diterpenoid glyscosides, stevioside and rebaudioside A. Although widely used in Japan and Brazil, most other countries do not allow the use of Stevia as a food additive. In the US, it can only be labelled as a dietary supplement. Many companies are now focusing on isolation of each of the individual diterpenoid glycosides, not just stevioside and rebaudioside A, for use in products as a low-calorie sugar alternative. Current HPLC methods for the determination of diterpenoid glycosides have been on NH₂-columns under isocratic conditions [1]. An improved HPLC method utilizing a Phenomenex Synergi Hydro-RP column has been developed on a gradient reverse-phase system of 0.1% trifluoroacetic acid in water and acetonitrile at 202 nm which overcomes the limitations of the reported NH₂ method with regards to separation of the minor components of Stevia. The method successfully separates rebaudiosides A, B, C, D and F, dulcoside A, isoteviol, steviol, steviol glucuronide, stevioside, steviolbioside, and several unidentified compounds.

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