Sensitive HILIC UHPLC-UV determination of steviol glycoside natural sweeteners

Significance of the Topic

Steviol glycosides are high-intensity natural sweeteners widely used in the food and beverage industry. Their intense sweetness, zero caloric content and increased regulatory scrutiny make robust, sensitive and reproducible analytical methods essential for quality control and food safety.

Study Objectives and Overview

This work demonstrates the transfer of a hydrophilic interaction liquid chromatography (HILIC) UHPLC-UV method for six steviol glycosides from a Thermo Scientific™ UltiMate™ 3000 system to a Vanquish™ Flex UHPLC system. Key goals include evaluating improvements in sensitivity and reproducibility achieved by new pump and autosampler technologies and the Thermo Scientific™ LightPipe™ flow cell for diode array detection.

Methodology and Instrumentation

Sample Preparation:

• Individual primary solutions of six glycosides prepared at 2 mg/mL (1 mg/mL for rebaudioside C) in mobile phase.
• Mixed standard spiking solution at 0.19 mg/mL per compound.

UHPLC Conditions:

• Column: Acclaim™ Trinity P1, 3 μm, 100 × 2.1 mm with guard cartridge.
• Mobile phase: 10 mM ammonium formate at pH 3.0 / acetonitrile (19:81, v/v).
• Flow rate: 0.30 mL/min; column and pre-heater at 40 °C; injection volume 5 μL; detection at 210 nm; 350 μL static mixer + 50 μL capillary.

Instrumentation

• UltiMate 3000 UHPLC: LPG-3400RS pump, WPS-3000RS autosampler, TCC-3000RS column oven, DAD-3000RS with 13 μL flow cell.
• Vanquish Flex UHPLC: Quaternary Pump F, Split Sampler FT, Column Compartment H, Active Pre-heater, Diode Array Detector HL with 2 μL LightPipe™ flow cell, Virtuoso™ Vial Identification System.

Main Results and Discussion

Both systems achieved baseline separation of six steviol glycosides within 10 minutes with comparable retention times, requiring no adjustment of dwell volumes. Peak widths at half height were under 0.2 min for most analytes on both platforms. The Vanquish Flex system exhibited up to an 80% reduction in %RSD of peak width (dulcoside A) and up to a 40% increase in peak height and area due to the LightPipe™ flow cell, demonstrating enhanced reproducibility and sensitivity.

Benefits and Practical Applications

• Improved sensitivity supports detection of low-level glycosides in complex matrices.
• Enhanced precision reduces re-validation requirements and QC failures.
• Seamless method transfer minimizes downtime and cost.
• HILIC separation on Acclaim Trinity P1 resolves highly polar, structurally similar glycosides.

Future Trends and Potential Applications

Integration of HILIC UHPLC with mass spectrometric or charged aerosol detection can further lower detection limits. Continued advances in flow cell design and pump precision, coupled with automation and data-driven analytics, will broaden applications to other polar analytes and real-time process monitoring.

Conclusion

The Vanquish Flex UHPLC system, paired with Acclaim Trinity P1 columns and LightPipe™ DAD flow cell technology, delivers superior sensitivity and reproducibility for HILIC analysis of steviol glycosides. This method transfer reduces operational costs and enhances data confidence in routine QA/QC workflows.

References

• Thermo Fisher Scientific. Application Note 21734: Sensitive HILIC UHPLC-UV determination of steviol glycoside natural sweeteners.