Ultra-HPLC method for quality and adulterant assessment of steviol glycosides sweeteners – Stevia rebaudiana and stevia products

Abstract

Stevia products are advertised as a zero-calorie sweetener. Glucose should not be an intrinsic component of this product, but it has been identified from some of stevia products in a preliminary study. An UHPLC-UV method was developed for the quantitative determination of glucose from stevia products. After stevia products reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP), PMP derivatives were analysed and glucose was found in seven out of 35 products in the range 0.3–91.5% (w/w). Two products, SPR-12 and SPR-27, showed remarkable amounts of glucose at 61.6% and 91.5%, respectively. In addition, an UHPLC-UV-evaporative light-scattering detector (ELSD) method was developed for the quantitative determination of rebaudioside A, stevioside, rebaudioside D, dulcoside A and steviolbioside from Stevia rebaudiana and related products. In a 12 min run, five steviol glycosides were baseline-separated. ELSD and ultraviolet (UV) detections showed comparable results. The LC methods were validated for linearity, repeatability, accuracy, limits of detection (LOD) and limits of quantification (LOQ). For steviol glycosides, the LODs and LOQs were found to be less than 10 and 30 μg ml^–1, respectively. The RSD for intra- and inter-day analyses was less than 2.5%, and the recovery was 90–94%. For PMP derivative of glucose, the LOD and LOQ were 0.01 and 0.05 μg ml^–1, respectively. Repeatability (RSD) was less than 2.6%; recovery was 98.6–101.7%. The methods are useful for the identification, quality assurance, and adulterant assessment of S. rebaudiana and steviol glycosides sweeteners (stevia products).

Graphical Abstract

Keywords:

• Stevia rebaudiana
• stevia products
• steviol glycosides
• glucose
• UHPLC-UV-ELSD

Acknowledgements

The authors thank Dr Jon Parcher, Research Professor Emeritus at the University of Mississippi, for providing support and valuable suggestions on the manuscript.

Additional information

Funding

This research was supported in part by the ‘Science Based Authentication of Dietary Supplements’ funded by the US Food and Drug Administration (USFDA) [grant number 1U01FD004246-003]; and by the United States Department of Agriculture (USDA), Agricultural Research Service [Specific Cooperative Agreement No. 58-6408-02-1-162-04].