EUSTAS Protocol and Round-Robin testing of steviol glycosides by an internal standard method

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, changes in sensitivity of the detector, etc. Moreover, use of an internal standard allows for the correction of losses due to sample clean-up of complex samples (e.g., after extraction of foods). An ideal IS is a compound with properties very similar to, and that behaves in a similar way to the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), should the IS be well separated from the compounds of the mixture to be analysed. We synthesized 19-O-beta-D-galactopyranosy1-13-O-beta-D-glucopyranosyl-steviol as an IS. The results show that the analyses of SVglys using an IS are very much simplified. The possible errors are much reduced. The inter-laboratory RSD for the analysis of all the SVglys present was about 1.75 %, which is much better than can be obtained by an external standard method. This value might still decrease after improvement of peak resolution and peak integration techniques in some laboratories. Our method made it possible to inject 5 x more of the same sample vials, resulting in a more accurate measurement of the small peaks, enhancing overall accuracy. Besides the analysis of SVglys, also the amount of SVeqs is given, expressed on a dry and wet wt. basis. The IS method is likely to become the method of choice for the whole Stevia industry.