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Improvement of the Assay Method for steviol Glycosides in the JECFA Specifications

American Journal of Analytical Chemistry 2013年第4期4卷 190-196页

作者： Atsuko Tada Kyoko Ishizuki Junichi Iwamura Hirohisa Mikami Yoshiko Hirao Isao Fujita Takeshi Yamazaki Hiroshi Akiyama Yoko Kawamura Laboratory of Creative Science Co. Ltd. Misonocho Japan Morita Kagaku Kogyo Co. Ltd. Osaka Japan National Institute of Health Sciences Tokyo Japan Shimadzu Corporation Kyoto Japan

steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.

关键词： steviol Glycosides Stevioside Rebaudioside A Reversed-Phase HPLC JECFA Specifications

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

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American Journal of Analytical Chemistry 2018年第11期9卷 547-559页

作者： Jan M. C. Geuns Tom Struyf Stijn Ceunen Gert Steurs Wim De Borggraeve Laboratory of Functional Biology KU Leuven Leuven Belgium Division of Molecular Design and Synthesis KU Leuven Leuven Belgium

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 (

关键词： steviol Glycosides Internal Standard Method Validation Quantification Purification

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Simple extraction and membrane purification process in isolation of steviosides with improved organoleptic activity

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Advances in Bioscience and Biotechnology 2012年第4期3卷 327-335页

作者： Adari Bhaskar Rao Ernala Prasad Goka Roopa Sundergopal Sridhar Yerrapragada Venkata Lakshmi Ravikumar Chemical Engineering Division Indian Institute of Chemical Technology Hyderabad India Chemical Engineering Division Indian Institute of Chemical Technology Hyderabad-7 India Organic Chemistry Division Indian Institute of Chemical Technology Hyderabad-7 India

In recent years there has been increasing demand for natural non-nutritive high intensity sweeteners with low-calorie value as an alternative to sucrose. Extracts of the leaves of Stevia rebaudiana (Bertoni), have been known for their sweet taste. Steviosides and rebaudioside-A are the two major diterpenoid glucosides components present in the leaf extracts of the stevia, these glycosides are 300 times sweeter than sugar and also exhibits wide therapeutic activity. The conventional methods of isolation of steviosides involve long extraction and purification procedures;therefore optimization of product yields is a challenging problem. The present study, establishes a new improvised process of extraction of steviosides from the stevia leaves in which the dry treated leaves were grounded, defatted, and extracted through pressurized hot water extractor (PHWE), followed by purification and concentration of the sweet glycosides through ultra (UF) and nano (NF) membrane filtration in obtaining high (98.2%) purity steviosides. This process established “green” method for isolation of high quality steviol glycosides, with improved final yield is 10.1% from 11% of crude leaf extract and observed the improved organoleptic and biological activity (antioxidant). Thus the method confirms a simple, inexpensive and eco-friendly process in obtaining pure steviosides.

关键词： steviol Glycosides Membrane-Filtration Natural-Sweetener Antioxidant In-Expensive

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