Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method

Jan M C Geuns,Wim De Borggraeve,Gert Steurs,Tom Struyf,Stijn Ceunen

doi:10.4236/ajac.2018.911040

Abstract

The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.

Highlights

Steviol glycosides (SVglys), the sweet diterpene glycosides found in Stevia rebaudiana Bertoni leaves, have been widely used as intense sweeteners
The LC-MS data (Figure 2) contain clear signals in the ESI-spectrum for the molecular ion at m/z 641.3 [M − H]− and a formic acid (FA) adduct at m/z 687.3 [M + FA − H]− (FA was added to the eluent)
An internal standard (IS) could be synthesised that behaved like steviol glycosides (SVglys) in all the steps of handling and is well separated from the known SVglys

Summary

Introduction

Steviol glycosides (SVglys), the sweet diterpene glycosides found in Stevia rebaudiana Bertoni leaves, have been widely used as intense sweeteners. In several countries their use is allowed in general food (China, Brazil, India, Japan, ...) or as a food additive (Australia, New Zealand, USA, EU). NH2 columns give a good separation, they have poor reproducibility and are not practical [7]. C18 columns are more robust but may give poor resolution. This can be solved by using two columns in series [10]. JECFA recommended the use of a C18 column [3]

Methods

Results

Conclusion