TITLE:
Improvement of the Assay Method for Steviol Glycosides in the JECFA Specifications
AUTHORS:
Atsuko Tada, Kyoko Ishizuki, Junichi Iwamura, Hirohisa Mikami, Yoshiko Hirao, Isao Fujita, Takeshi Yamazaki, Hiroshi Akiyama, Yoko Kawamura
KEYWORDS:
Steviol Glycosides; Stevioside; Rebaudioside A; Reversed-Phase HPLC; JECFA Specifications
JOURNAL NAME:
American Journal of Analytical Chemistry,
Vol.4 No.4,
April
29,
2013
ABSTRACT: 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.