Ultrahigh-Performance Supercritical Fluid Chromatography−Multimodal Ionization−Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts

Importance of the Topic

The chemical diversity of plant extracts spans more than 200 000 metabolites varying from nonpolar volatile terpenes to highly polar flavonoids and phenolic acids. Traditional analysis often combines gas chromatography for volatiles and liquid chromatography for polar compounds. This workflow is labor-intensive, time-consuming, and requires multiple instruments. Developing a unified high-throughput method that handles the full polarity range in a single platform promises significant gains in efficiency, reproducibility, and throughput for natural product research, quality control, and metabolomics.

Objectives and Study Overview

This study aimed to establish a universal two-injection approach using ultrahigh-performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPSFC-MS/MS) to cover both lipophilic terpenes and polar flavonoids/phenolic acids in complex plant extracts without manual intervention. Key goals included optimized column selection, gradient design, ionization source evaluation, and quantitative validation. The method was demonstrated on Eucalyptus sp. extracts obtained by supercritical fluid extraction (SFE).

Methodology and Instrumentation

The workflow comprised two UHPSFC injections on a single Acquity UPC2 system equipped with:

• Column manager for simultaneous use of two columns
• Porous graphitic carbon (PGC, 3.0×150 mm, 2.7 μm) for volatile terpenes
• Diol column (3.0×50 mm, 1.7 μm) for flavonoids and phenolic/terpenoic acids
• Binary pumps for CO₂ and organic modifier delivery, back-pressure regulator and column thermostat
• Triple quadrupole Xevo TQ-XS MS with interchangeable ESI, APCI, ESCi, and UniSpray sources

Design of experiments (DoE) optimized mobile phase additives (ammonia, water, formic acid), makeup solvent composition, flow rates, and MS parameters (cone voltage, desolvation gas, temperature) for each ion source.

Main Results and Discussion

Column screening revealed only PGC retained nonpolar terpenes with sufficient resolution. A longer 150 mm PGC column at 60 °C and 22.75 MPa achieved baseline separation of 17 isobaric terpenes within 7.5 min. A 50 mm diol column with 5% water in MeOH gradient and pressure programming separated 23 polar analytes in 15.5 min. ESCi multimodal ionization allowed detection of both lipophilic terpenes (via APCI mode) and polar flavonoids (via ESI mode) in a single run without hardware changes. Quantitative performance delivered lower limits of quantification of 0.01–1 ng/mL for terpenes and 0.5–20 ng/mL for flavonoids, with retention time RSD <1.5% and area RSD <10%. Application to Eucalyptus sp. SFE extracts revealed >90% terpenoic acids, ~9% flavonoids/phenolics, and ~0.4% terpenes by relative abundance.

Benefits and Practical Applications

The two-injection UHPSFC-MS/MS approach streamlines complex plant extract analysis by:

• Reducing the need for separate GC and LC runs
• Minimizing sample handling and manual intervention
• Shortening total analysis time (<25 min total chromatographic time)
• Providing robust quantification across wide polarity ranges

This method is suitable for metabolomics, pharmaceutical profiling, quality control in herbal products, and industrial analytics.

Future Trends and Possibilities

Advances could include:

• Development of universal stationary phases to combine polar and nonpolar separations in one injection
• Integration of direct GC-MS hyphenation for fully automated volatile analysis
• Enhanced multimodal ionization combining photoionization for nonpolar aromatics
• Application to 2D-SFC workflows for ultra-complex mixtures

Continued hardware and column innovations will further expand UHPSFC’s universal applicability.

Conclusion

A novel UHPSFC-MS/MS method employing two injections on a single instrument was developed to analyze small lipophilic terpenes and polar flavonoids/phenolic acids in complex plant extracts. The combination of PGC and diol columns with ESCi ionization delivered high throughput, sensitivity, and robustness, addressing a critical analytical challenge in natural product research. This approach offers a unified, efficient alternative to traditional GC and LC workflows.

References

1. Plachká K, Pilarová V, Kosturko Š, Škop J, Svec F, Nováková L. Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts. Anal. Chem. 2024, 96, 2840–2848.
2. Ganzera M, Zwerger M. Complex analysis of plant extracts. Trends Anal. Chem. 2021, 145, 116463.
3. Si-Hung L, Bamba T. Unified chromatography–hydrophilic interaction liquid chromatography for metabolomic analysis. Anal. Chim. Acta 2023, 1240, 340741.