CHAPTER 4: Analysis of Steviol Glycosides

In this chapter actual trends in sample preparation, determination, detection and quantification of steviol glycosides were reviewed beginning with the first official method for the analysis of steviol glycosides published in 1993. Before analysis, various sample preparation techniques have to be applied depending on the type of sample. Leaves of Stevia rebaudiana are traditionally extracted with hot water but it was shown that the application of ultrasound- and microwave-assisted extraction can shorten the extraction time and improve the yield. Food and other complex matrices often need a further clean-up, e.g. solid phase extraction (SPE) before determination. The method of choice to quantify steviol glycoside contents is high performance liquid chromatography (HPLC) in combination with different column types like Amino, HILIC, reversed phase (RP) and further, more specific phases. Actually, RP columns are preferred in the official protocols. Detection can be done by UV-detectors or more sensitive and selective by mass spectrometry. Steviol glycosides can properly be distinguished by their typical fragmentation pattern. Alternative detectors like evaporative light scattering detector (ELSD) or a charged aerosol detector (CAD) also play a role in the analysis of the sweeteners. Techniques like thin-layer-chromatography, capillary electrophoresis (CE), ¹H-NMR spectroscopy and near-infrared reflectance spectroscopy (NIRS) are often applied for screening analyses. An alternative approach is to determine steviol glycosides as a sum parameter via their aglycon steviol.