Fractionation of Anthocyanins by Preparative LC-MS System

Significance of the Topic

Anthocyanins are natural flavonoid pigments responsible for red to blue coloration in plant tissues. Beyond their application as food colorants, they exhibit potent antioxidant activity and potential health benefits. Isolating individual anthocyanin species from complex botanical extracts is crucial to understand their distinct biological functions and to support quality control in nutraceutical and food industries.

Objectives and Study Overview

This study demonstrates a preparative liquid chromatography–mass spectrometry (LC-MS) approach to fractionate four major anthocyanins from blackcurrant extract. The primary goals are to achieve high purity isolations of each compound and to illustrate how MS-triggered collection parameters can be tuned to favor either purity or recovery.

Methodology

Blackcurrant capsule extract (0.5 g) was dissolved, sonicated, filtered, and injected into the preparative LC-MS system. Fractions were collected based on positive-mode electrospray ionization signals at target m/z values of 465, 611, 449, and 595, corresponding to delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside respectively. Two collection strategies were compared:

• High purity mode: elevated signal threshold to minimize co-eluting impurities.
• High recovery mode: single-ion monitoring at m/z 611 with a lower threshold to capture the full peak of delphinidin-3-rutinoside.

Used Instrumentation

• Preparative LC-MS System: Shimadzu prepLCMS-2010EV with Gemini C18 Axia column (21.2 mm × 50 mm, 5 µm).
• Analytical UFLC-MS: Shimadzu Prominence UFLC coupled to LCMS-2010EV with XR-ODS column (2.0 mm × 50 mm).
• Mobile phases: Water and acetonitrile with 0.1 % trifluoroacetic acid. Methanol makeup flow for preparative runs.

Main Results and Discussion

Preparative fractionation yielded four distinct fractions with purities between 92 % and 99 % as confirmed by ultra-fast LC-MS analysis. High-purity mode produced clean isolations with minimal adjacent peaks. Alternatively, high-recovery mode captured full analyte peaks at the expense of minor co-elution, demonstrating the flexibility of MS-triggered collection. Chromatographic runs lasted 30–40 minutes for preparative separations and under 10 minutes for analytical verification.

Benefits and Practical Applications

The demonstrated workflow enables rapid isolation of structurally similar anthocyanins with tunable purity and yield. Such capability supports:

• Structural and functional studies of individual anthocyanins.
• Reference standard production for method validation.
• Quality control in plant extract manufacturing.

Future Trends and Potential Applications

Advancements in preparative LC-MS, including higher flow-rate columns, improved ion-triggering algorithms, and automated fraction recovery, will enhance throughput and scalability. Integration with real-time spectral deconvolution may further refine purity control. Such developments can broaden applications in natural product research, personalized nutrition, and pharmaceutical lead discovery.

Conclusion

This work highlights an effective preparative LC-MS strategy for isolating anthocyanins from blackcurrant extract. By adjusting MS signal thresholds, users can prioritize either compound purity or recovery, addressing diverse research and production needs. The approach offers a robust platform for isolating minor flavonoid constituents and generating high-quality analytical standards.

References

• Application News No. C60A: Fractionation of Anthocyanins by Preparative LC-MS System, Shimadzu Corporation.
• Application News No. L340A: Analysis of Anthocyanins by HPLC, Shimadzu Corporation.