Absolute Quantitation of Stevioside and Rebaudioside A in Commercial Standards by Quantitative NMR

Abstract

The extract produced from the leaves of Stevia rebaudiana Bertoni (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A (Fig. 1), and has long been used as a sweetener in Japan. In 2008, the specifications for "steviol glycosides" were established by the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA), and "stevia extracts" were approved as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA). Thereafter, the purified stevia extracts specified in JECFA and FDA became popular worldwide.According to the assay for steviol glycocides in the JECFA specifications, 1) the total content of nine types of steviol glycosides (Fig. 2) should not be less than 95%. In the assay method, rebaudioside A is quantified from the peak area on LC using the rebaudioside A standard, and each steviol glycoside except rebaudioside A is quantified from their respective peak areas and absorption coefficients using the stevioside standard as the quantification standard.Various commercial standard reagents of stevioside and rebaudioside A are available, although their purities are different and their exact purities are not indicated in the product literatures. Therefore, if the claimed values are employed in the quantitative analysis of a sample, the quantitated contents of the stevioside and rebaudioside A in the identical sample will be assigned different measured values according to the standard used for the quantification.Quantitative NMR (qNMR) using an internal standard with traceability to the International System of Units (SI units) has recently been used to determine the absolute purities of various chemical substances.2-7) The qNMR method permits the absolute quantification of a target compound without the need for a standard of that compound. In addition, it is rapid and easy. The compound is quantified from the ratios of the integral values of the proton signals of the internal standard and the target compound. In addition, various compounds can be quantified using the same internal standard.In this study, we utilized the qNMR method to determine the purity of stevioside and rebaudioside A in commercial standards. The knowledge of their absolute purities will increase the accuracy of other analytical methods that use standards, such as LC.

Digital NMR Profiles as Building Blocks: Assembling 1H Fingerprints of Steviol Glycosides

“…At least two previous reports have described the use of quantitative 1 H NMR to examine mixtures of steviol glycosides, 17,18 making a compelling case for the application of NMR spectroscopy as a complementary approach to conventional LC-UV and LC-MS methods.…”

Rebaudioside A inhibits pentylenetetrazol‐induced convulsions in rats

“…Reasons for its preference among convenience food manufacturers include high temperature‐ and pH‐stability [13]. Reb‐A and stevioside are steviol glycosides derived from the S. rebaudiana herb [12,14–16].…”

A Universal Quantitative 1H Nuclear Magnetic Resonance (qNMR) Method for Assessing the Purity of Dammarane‐type Ginsenosides

“…The characteristic of quantitative estimation without relying on target reference substances makes the qNMR technique especially suitable for purity tests of chemical compounds and natural products. There have been many articles published on qNMR for purity tests of agrochemicals (Al Deen et al , ; Wells et al , ), pharmaceutical chemicals (Liu and Hu, ) and natural products (Tada et al , ). All of these investigations indicate that the qNMR method can be a feasible protocol for purity tests of ginsenoside standards.…”