Separation of Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography
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Separation of Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography Benno F. Zimmermann · Ursula Woelwer-Rieck · Menelaos Papagiannopoulos
Received: 14 November 2010 / Accepted: 14 March 2011 / Published online: 13 April 2011 © Springer Science+Business Media, LLC 2011
Abstract Stevia rebaudiana Bertoni contains several steviol glycosides with sweet flavour. They all are sweeter than sucrose (up to factor 450). The various steviol glycosides are difficult to separate by reversedphase chromatography. In this paper, five different hydrophilic liquid interaction chromatography columns are characterized using isocratic elution (5–20% water in acetonitrile with buffer or formic acid). Separation of the steviol glycosides is possible with all but one of the tested columns, but the robustness of the separation against changes of buffer concentration and percentage of water differ. Aqueous percentage and ion strength of the eluent are the main factors to be optimized in method development. Keywords Stevia rebaudiana · Hydrophilic liquid interaction chromatography · Steviol glycosides · HPLC · Mass spectrometry Introduction Stevia rebaudiana Bertoni (Asteraceae) contains several steviol glycosides with sweet flavour. They all B. F. Zimmermann · U. Woelwer-Rieck · M. Papagiannopoulos Institute of Nutrition and Food Sciences, University of Bonn, Endenicher Allee 11–13, 53115 Bonn, Germany B. F. Zimmermann (B) Institut Prof. Dr. Kurz GmbH, Eupener Str. 161, 50933 Cologne, Germany e-mail: [email protected] Present Address: M. Papagiannopoulos Corporate Analytical Services–Development, Weleda AG, Möhlerstraße 3, 73525 Schwäbisch Gmünd, Germany
are sweeter than sucrose (up to factor 450; Dacome et al. 2005). Prakash et al. (2008) composed a table showing the structures of ten steviol glycosides and their molecular masses. Dacome et al. (2005) reported in a table the relative sweetening power of the same steviol glycosides. In 2008, the US Food and Drug Adminstration accepted several highly purified steviol glycosides (containing >95% rebaudioside A) as generally recognized as safe (FDA 2008a, b, c). In April 2010, the European Food Safety Authority (EFSA) stated the safety of steviol glycosides (mixtures of steviol glycosides that comprise not less than 95% of stevioside and/or rebaudioside A) considering them as not genotoxic, carcinogenic, nor associated with any reproductive/developmental toxicity (EFSA 2010). Thus, one can expect that such highly purified extracts will be allowed to be marketed in Europe. For separation of steviol glycosides, highperformance liquid chromatography (HPLC) is the method of choice; other methods as capillary electrophoresis and high-performance thin-layer chromatography have been proposed as reviewed recently by Madan et al. (2010), but there are much more scientific articles dealing with HPLC. Reversedphase (RP) columns show poor selectivity with regard to the separation of stevioside and rebaudioside A (Geuns 2010; Clos et al. 2008). In both cited methods, separation t
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