Oh so sweet - Quantification of steviol glycosides in Stevia samples with RP-HPLC

Importance of the Topic

Steviol glycosides from Stevia rebaudiana serve as high-intensity natural sweeteners approved in the EU since 2011. Their substantially greater sweetness compared to sucrose, combined with negligible caloric impact and suitability for diabetic and weight-management diets, drives the need for robust analytical methods to ensure product quality and authenticity.

Objectives and Study Overview

This study aimed to develop and validate a rapid reversed-phase HPLC method for simultaneous quantification of six major steviol glycosides (rebaudioside A, stevioside, rebaudioside C, dulcoside A, rebaudioside B and steviolbioside) in both raw leaf extracts and processed sweetener formulations. Emphasis was placed on achieving baseline separation in under 10 minutes to support fast quality control workflows.

Used Methodology and Instrumentation

Solid-liquid extraction of dried leaves and dissolution of commercial sweetener powders were followed by filtration and online sample purification via SPE. Chromatographic separation employed a binary gradient of water (A) and acetonitrile (B) at 1.2 mL/min on a C18 column (250 × 4.6 mm, 5 µm) maintained at 40 °C. Detection was performed at 210 nm with simultaneous UV spectral recording from 200 to 700 nm.

Main Results and Discussion

Calibration at 0.1 mg/mL per analyte yielded baseline separation of all six compounds, including the critical rebaudioside A/stevioside pair. Analysis of dried leaf extract and commercial sweetener revealed accurate quantification with relative mass fractions aligning with expectations for raw material but exposing additional glycosides in the sweetener beyond declared rebaudioside A. 3D PDA data confirmed absence of co-eluting interferences.

Benefits and Practical Applications of the Method

• Fast throughput: complete run including equilibration in 12 min
• High resolution and baseline separation of structurally similar compounds
• Robustness confirmed by DryLab gradient/temperature simulation
• Improved detection limits via SPE cleanup

Future Trends and Potential Applications

Integration of UHPLC for even shorter cycle times, coupling with mass spectrometry for enhanced selectivity, and application of automated sample preparation will further streamline steviol glycoside analysis. Data-processing advances and green solvent alternatives are expected to optimize sustainability and regulatory compliance.

Conclusion

The presented RP-HPLC method provides a reliable, rapid, and precise approach for comprehensive profiling of steviol glycosides in diverse Stevia matrices, supporting industry-level quality assurance and product development.

References

• Teresa Brandes, Marion Raters, Reinhard Matissek. Steviolglycoside in Süßwaren – Entwicklung und Validierung einer Analysenmethode mittels HPLC-UV. DLR. 2013.
• René Borstel. Fast online SPE purification of Stevia plant extracts. Application Note VFD0093N. 2011.
• Venkata Sai Prakash Chaturvedula, Julian Zamora. Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni. Food and Nutrition Sciences. 2014;5:1711–1716.