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

作     者：Atsuko Tada Kyoko Ishizuki Junichi Iwamura Hirohisa Mikami Yoshiko Hirao Isao Fujita Takeshi Yamazaki Hiroshi Akiyama Yoko Kawamura 

作者机构：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 

出 版 物：《American Journal of Analytical Chemistry》 (美国分析化学（英文）)

年 卷 期：2013年第4卷第4期

页      面：190-196页

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

主　　题：Steviol Glycosides Stevioside Rebaudioside A Reversed-Phase HPLC JECFA Specifications 

摘      要：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.