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

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.

Determination of Rebaudioside A and Stevioside in Leaves of <i>S. rebaudiana</i> Bertoni Grown in México by a Validated HPLC Method

“…The precision for retention time was ≤1.93 %RSD in all cases, while for peak area was ≤6.27% (Table 3). In this regard, the %RSD of peak area found is higher than that reported for rebaudioside A (0.70%) or stevioside (0.41%) using a similar chromatographic method [19], that may be attributed to the purity of the standards (99%) used by Tada et al (2011); however given the %RSD obtained here, the method can be considered precise to quantify both rebaudioside A and stevioside.…”

“…Considering that the results are only comparable with what has been reported for S. rebaudiana grown in Mexico, this discrepancy could be due to the effect of the growing season or harvest (related to solar radiation and rain precipitation) or the use of a different analytical method. In fact, JECFA method [21] used in this work involves a more stable column compare to amino bonded column [19], lower retention times for rebaudioside A and stevioside, and a lower proportion of acetonitrile in the mobile phase than the method described for amino bonded columns. Taking these data together, the content of major glycosides found here are from a validated method, which involves less time and use of moderately toxic substance as it is the acetonitrile.…”

Rapid HPLC Method for Determination of Rebaudioside D in Leaves of <i>Stevia rebaudiana</i> Bertoni Grown in the Southeast of México

“…The precision for retention time was ≤0.79% RSD in all cases, whereas the relative standard deviation for peak area was ≤2.79% (Table 3). In this regard, the % RSD of peak area found is slightly higher than those reported for the major glycosides Rebaudioside A (0.70%) and stevioside (0.41%) using a similar chromatographic method [18], but less than 10% which is the value generally regarded as appropriate and therefore it can be considered that this method is precise to quantify rebaudioside D.…”

Effect of different drying methods on the composition of steviol glycosides in Stevia rebaudiana Bertoni leaves

“…-different analytical techniques (Tada et al, 2013;Wöelwer-Rieck et al, 2010). Therefore, the results presented in this paper highlight an additional factor to be considered: the method of drying, taking into account that the same variety of Stevia rebaudiana Bertoni, extraction condition, and analytical technique were used.…”