Rapid analysis of natural sweeteners found in food and beverages using an advanced UHPLC system

Significance of the topic

Natural steviol glycosides derived from Stevia rebaudiana are increasingly used as low-calorie sweeteners in food and beverages. Their high sweetness intensity, coupled with potential health benefits, has driven demand for robust analytical methods capable of rapid, reliable quantification in quality control and research settings.

Objectives and overview of the study

This study aimed to develop and validate a fast UHPLC-HILIC method for baseline separation of six major steviol glycosides (dulcoside A, stevioside, rebaudioside A, B, C and steviobioside) using a Thermo Scientific Vanquish Flex system equipped with a Syncronis HILIC column.

Methodology and applied instrumentation

Sample preparation involved dissolving standards at 2.0 mg/mL (1.0 mg/mL for rebaudioside C) and working solutions at 0.2 mg/mL in mobile phase. Detailed UHPLC conditions and key instrumentation components included:

• System: Vanquish Flex UHPLC with quaternary pump, Split Sampler FT, column compartment and Diode Array Detector featuring LightPipe flow cell (10 mm).
• Column: Thermo Scientific Syncronis HILIC, 100 × 2.1 mm, 1.7 µm.
• Mobile phase: A = 10 mM ammonium formate, pH 3.0; B = acetonitrile; isocratic 15% A/85% B.
• Flow rate: 0.8 mL/min; column temperature: 40 °C; injection volume: 2 µL; detection at 210 nm.
• Consumables: LC-MS grade water, acetonitrile, formic acid, ammonium formate, nylon filters, labeled vials.

Main results and discussion

Column screening showed optimal retention and peak shape on Syncronis HILIC versus alternative HILIC phases. Small adjustments in buffer concentration and organic ratio (±1%) significantly affected retention and resolution. A 350 µL static mixer minimized baseline noise. Increasing column temperature from 25 to 45 °C improved symmetry and reduced retention times; 40 °C was selected as a compromise. Flow rate optimization (0.5–0.8 mL/min) maintained full resolution within a 2 min run. The method achieved retention time precision below 0.1% RSD over 24 replicates. Application to commercial samples revealed distinct glycoside profiles among tested brands.

Benefits and practical applications

• High throughput: 12-fold faster than legacy methods, enabling >30 sample runs per hour.
• Cost reduction: 8-fold lower mobile phase consumption per analysis.
• Reproducibility: excellent retention stability and peak precision.
• Versatility: suitable for product screening and routine QC of natural sweeteners.

Future trends and possibilities

Future enhancements may include coupling to mass spectrometry for increased selectivity, miniaturized HILIC formats for further speed gains, automated sample preparation workflows, and extension of the approach to emerging natural sweetener classes.

Conclusion

A rapid, robust UHPLC-HILIC method for six steviol glycosides has been established, delivering high resolution, excellent reproducibility and significant reductions in analysis time and solvent usage, meeting the demands of modern analytical laboratories.

References

• Thermo Scientific HILIC Separations Technical Guide, TG-21003-HILIC-Separations.
• Thermo Scientific Technical Note 108: Reliable solvent mixing in UHPLC, LPN2851-EN.
• Thermo Scientific Application Note 241: Determination of steviol glycosides by HPLC with UV and ELS detections, AN70235-EN.