Sensitive UHPLC-UV determination of anthocyanins using innovative active eluent pre-heating technology

Importance of the Topic

Anthocyanins are flavonoid pigments found in fruits and flowers and are valued for their vibrant colors and potent antioxidant activity. Accurate quantification of anthocyanins is critical in food safety, nutraceutical development, and quality control workflows. Method robustness and reproducibility across different UHPLC platforms are essential to minimize re-validation costs and ensure consistent data confidence.

Goal and Study Overview

This study aimed to evaluate the transfer of an anthocyanin separation method from the Thermo Scientific UltiMate 3000 UHPLC system to the Vanquish Flex UHPLC system. Key objectives included comparing retention time precision, peak width, and peak area reproducibility using a single Acclaim RSLC 120 C18 column without altering the chromatographic method.

Methodology and Instrumentation

The chromatographic method employed an Acclaim RSLC 120 C18 2.2 µm column (150 mm × 2.1 mm) at 30 °C under a binary gradient of acetonitrile/formic acid/water at a flow rate of 0.475 mL/min. Six anthocyanins (delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, delphinidin-3-glucoside, pelargonidin-3,5-diglucoside, cyanidin-3-glucoside, and pelargonidin-3-glucoside) were prepared at 10 µg/mL each. Instrumentation included:

• Thermo Scientific Vanquish Flex UHPLC with active eluent pre-heater
• Thermo Scientific UltiMate 3000 UHPLC system
• Thermo Scientific diode array detectors set to 540 nm
• Thermo Scientific Virtuoso vial identification system

Main Results and Discussion

Both systems achieved baseline separation of the six anthocyanins within four minutes. The Vanquish Flex system equipped with active pre-heating demonstrated improved retention time precision (lower %RSD) and reduced peak width variability compared to the UltiMate 3000. Method transfer required no adjustment to dwell volume or gradient profile, confirming direct comparability of retention times.

Benefits and Practical Applications

The validated method transfer eliminates the need for re-validation, saving time and resources. Active eluent pre-heating enhances method reproducibility and reliability, supporting rigorous QA/QC protocols in environmental, food safety, and nutraceutical analyses.

Future Trends and Opportunities

Eluent temperature control is poised to benefit broader UHPLC applications by mitigating thermal mismatch and viscous heating effects at high pressures. This innovation enables high-throughput workflows, seamless method transfers, and increased confidence in quantitative results across laboratories and industries.

Conclusion

The study confirms the Acclaim RSLC 120 C18 column’s capability for rapid anthocyanin profiling and demonstrates a seamless, no-modification method transfer between UHPLC platforms. Active eluent pre-heating further enhances precision and operational efficiency.

Reference

• Thermo Fisher Scientific. Application Note 21731: Sensitive UHPLC-UV determination of anthocyanins using innovative active eluent pre-heating technology.