Improving Productivity in Purifying Antroquinonol Using UltraPerformance Convergence Chromatography (UPC2) and Preparative Supercritical Fluid Chromatography (Prep SFC)

Importance of the Topic

The isolation and purification of natural product leads such as antroquinonol is a critical step for drug discovery and nutraceutical development. Antroquinonol, a bioactive ubiquinone derivative from Antrodia camphorata, requires high purity (>99%) for medicinal research. Conventional reversed-phase liquid chromatography (RPLC) often limits resolution, loading capacity, and throughput when separating structurally similar analogs under preparative conditions.

Study Goals and Overview

This study compares two chromatographic workflows for purifying antroquinonol from a 98% raw extract:

• An RPLC-based preparative HPLC method.
• A supercritical fluid chromatography (SFC) approach combining analytical UltraPerformance Convergence Chromatography (UPC2) and preparative SFC.

Key objectives include evaluating resolution, elution order, sample loading, productivity, and solvent consumption to achieve >99% product purity efficiently.

Methodology and Instrumentation

Analytical characterization and preparative experiments were conducted using Waters platforms:

• UPLC H-Class with SQ Detector 2 (ESI+ detection).
• ACQUITY UPC 2 with TQD mass spectrometer (APCI+ and ESI+ detection).
• AutoPurification LC system with 3100 MS detector for preparative HPLC.
• Prep 100q SFC system with 3100 MS detector for preparative SFC.

Columns and phases:

• ACQUITY UPLC HSS T3, Atlantis T3 C18, ACQUITY UPLC BEH C18 for RPLC.
• UPC2 2-EP and Viridis Silica 2-EP for UPC2 and SFC.

Mobile phases included water/methanol or methanol/isopropanol for LC and CO₂ with isopropanol modifier for UPC2/SFC. Detailed gradients, flow rates, backpressures, temperatures, and injection volumes were optimized to maximize resolution and loading capacity.

Main Results and Discussion

In RPLC, antroquinonol and its demethoxylated impurity were baseline separated only at low injection volumes (≤10 µL). Scaling to semi-prep conditions projected a maximum of ~3.4 mg per injection, with resolution deteriorating at higher loads. UPC2 provided superior analytical resolution and reversed the elution order, allowing the target to elute before the impurity. This orthogonal selectivity enabled a loading increase to 12 mg (600 µL at 20 mg/mL) per injection on a 19 × 100 mm column and reduced run time from 20 to 8 min.

Benefits and Practical Applications

The combined UPC2 and preparative SFC workflow delivered:

• A ninefold increase in purification productivity (from 0.25 g to 2.25 g per 24 h).
• A 77% reduction in organic solvent usage.
• Enhanced selectivity for closely related analogs and isomers.
• Shorter cycle times and higher sample throughput.

This approach is particularly advantageous in pharmaceutical, nutraceutical, and traditional medicine settings where high-purity natural products are required at scale.

Future Trends and Opportunities

Emerging directions include:

• Development of novel stationary phases to further improve selectivity.
• Integration of automated fraction collection and real-time MS monitoring.
• Expansion of green chromatography techniques to minimize CO₂ and organic solvent footprints.
• Applications to a broader range of natural product classes and complex mixtures.

Conclusion

The integration of analytical UPC2 with preparative SFC offers a robust and sustainable alternative to RPLC for antroquinonol purification. By improving resolution, reversing elution order, and increasing loading capacity, this workflow significantly enhances productivity and reduces solvent consumption, addressing key bottlenecks in natural product drug discovery.

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