Article

Separation of Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography

Institut Prof. Dr. Kurz GmbH, Eupener Str. 161, 50933 Cologne, Germany
Food Analytical Methods (Impact Factor: 1.8). 04/2012; 5(2):266-271. DOI: 10.1007/s12161-011-9229-x

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 reversed-phase 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

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    • "[33] [34] The growing interest in the use of steviol glycosides has motivated research aimed to characterize and separate these sweetening agents. [35] [36] [37] [38] For separation, high-performance liquid chromatography (HPLC) using amino, [39] [40] [41] C18 [42] [43] and HILIC columns [44] [45] is considered the method of choice. Detection has been performed by optical spectroscopy [46] and MS, [47] [48] [49] [50] including direct approaches using desorption/ionization ambient MS techniques. "
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    ABSTRACT: Traveling wave ion mobility mass spectrometry (TWIM-MS) is shown to be able to separate and characterize several isomeric forms of diterpene glycosides stevioside (Stv) and rebaudioside A (RebA) that are cationized by Na(+) and K(+) at different sites. Determination and characterization of these coexisting isomeric species, herein termed catiomers, arising from cationization at different and highly competitive coordinating sites, is particularly challenging for glycosides. To achieve this goal, the advantage of using CO2 as a more massive and polarizable drift gas, over N2 , was demonstrated. Post-TWIM-MS/MS experiments were used to confirm the separation. Optimization of the possible geometries and cross-sectional calculations for mobility peak assignments were also performed. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
    Journal of Mass Spectrometry 02/2015; 50(2). DOI:10.1002/jms.3532 · 2.71 Impact Factor
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    • "Therefore, hydrophilic interaction liquid chromatography (HILIC) columns have been successfully applied to overcome these problems. [12] [13] After separation, steviol glycosides can be detected by UV and MS, with the latter being more sensitive. [11] [14] [15] Single MS detection of steviol glycosides is more sensitive than tandem mass spectrometry; thus, single MS is the preferred method for quantitation. "
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    ABSTRACT: Stevia rebaudiana contains several steviol glycosides that have a sweet flavor. They are up to 450 times sweeter than sucrose, but some have an undesirable aftertaste. Up to 2010, ten different steviol glycosides have been described from the leaves or purified extracts of S. rebaudiana. In this paper, the tandem mass spectrometric fragmentation patterns of these ten compounds are compiled, along with a scheme for structural elucidation. This scheme is then applied to 12 steviol glycosides that have not yet been described. The proposed structures of five steviol glycosides have been confirmed by other authors.
    Rapid Communications in Mass Spectrometry 06/2011; 25(11):1575-82. DOI:10.1002/rcm.5024 · 2.64 Impact Factor
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    ABSTRACT: In addition to their widely recognized use as dietary supplement ingredients, plant-derived compounds are increasingly used as natural sweeteners. The search for nonnutritive sweeteners has been stimulated over the last 20-30 years by concern over demonstrated or suspected relationships between consumption of sucrose and high-fructose corn syrups and a variety of health-related conditions. In the USA, there is increased use of plant extracts known to contain highly sweet terpenoids. Purified extracts of Stevia rebaudiana (Bertoni) containing the diterpene glycosides stevioside and rebaudioside A are popular as sweeteners and are also used as dietary supplements, and soft drinks and nutritional and energy shakes incorporating extracts of Siraitia grosvenorii (Swingle) fruits containing sweet triterpene glycosides such as mogroside V are also on the market. Here, we review recent studies on these two important sources of noncaloric natural sweeteners, including analytical methods used to identify and quantify specific constituents and structural features relating to their sweetness. We also review the generally recognized as safe status of specific components and their status with respect to review by the Joint FAO/WHO Expert Committee on Food Additives.
    Analytical and Bioanalytical Chemistry 01/2013; 405(13). DOI:10.1007/s00216-012-6693-0 · 3.58 Impact Factor
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