Determination of six steviol glycosides using reversed phased HPLC and online SPE

Importance of Determining Steviol Glycosides

Steviol glycosides from Stevia rebaudiana serve as natural, non-caloric sweeteners with approximately 400-fold higher sweetness than sucrose. Accurate quantification of these compounds is critical for quality control in food and beverage production, regulatory compliance, and ensuring consistency of stevia-based products.

Objectives and Study Overview

This work aimed to integrate online solid-phase extraction (SPE) with reversed-phase high-performance liquid chromatography (HPLC) and UV detection to achieve rapid, automated analysis of six key steviol glycosides (rebaudioside A, stevioside, rebaudioside C, dulcoside A, rebaudioside B and steviolbioside). Emphasis was placed on minimizing manual sample prep, reducing matrix effects, and maximizing throughput.

Methodology and Instrumentation

The method employs a water/acetonitrile gradient on a C18 analytical column with UV detection at 210 nm. Crude extracts of dried stevia leaves were prepared according to established protocols and spiked to generate a five-point calibration (0.01–0.15 mg/mL per analyte). Online SPE was performed using a small C18 cartridge to remove matrix components automatically. A timed valve-switching sequence directs the cleaned eluate onto the main column after a 7 min wash, resulting in a total run time of 15 min.

• KNAUER AZURA Online SPE System with P 6.1L pump, AS 6.1L autosampler, CT 2.1L column thermostat and DAD 2.1L detector
• AZURA ASM 2.1L SPE module with multi-position solvent selection and injection valves plus a P 4.1S feed pump
• Vertex Plus Eurospher II C18 columns (30 × 4.6 mm ID for SPE; 250 × 4.6 mm ID for analytical separation)

Main Results and Discussion

The combined SPE–HPLC method achieved baseline resolution of all six glycosides within 15 minutes per sample. Automated matrix reduction removed interfering components effectively, as monitored by flow-through profiles, and added only 3 minutes to the original 12 minute HPLC runtime. Reproducible calibration curves and peak shapes demonstrated the method’s robustness and suitability for routine analysis.

Benefits and Practical Applications

Incorporating online SPE offers multiple advantages for laboratory workflows:

• Substantial reduction in manual sample handling and associated variability
• Increased sample throughput for high-volume quality control testing
• Protection and extended lifetime of the analytical column by reducing matrix load

Future Trends and Potential Uses

Further developments may include coupling the online SPE-HPLC setup with mass spectrometric detection for enhanced sensitivity, expanding the approach to additional natural product analytes, and miniaturizing the SPE format to reduce solvent consumption and enable field-deployable analyses.

Conclusion

The described online SPE-HPLC method provides a fast, reliable, and largely automated solution for quantifying six steviol glycosides in Stevia extracts. By streamlining sample preparation and protecting analytical hardware, it meets the demands of modern food quality laboratories and supports consistent product evaluation.

Reference

• Samuel P, Ayoob KT, Magnuson BA et al. Stevia Leaf to Stevia Sweetener: Exploring Its Science, Benefits, and Future Potential. Journal of Nutrition. 2018;148(7):1186S–1205S.