ionKey/MS Ion Mobility: A New Approach to Authentication and Routine Screening of Ginsenoside Isomers in Functional Food Products

Importance of the Topic

Proper authentication and routine screening of ginsenoside isomers are essential for ensuring the quality, safety, and efficacy of functional food and nutraceutical products. Traditional chromatographic assays often struggle to resolve structurally similar saponins, leading to potential misidentification. Incorporating ion mobility mass spectrometry adds an orthogonal separation dimension based on size, shape, and charge, dramatically improving selectivity.

Objectives and Study Overview

This work evaluates a microflow chromatography platform coupled with ionKey/MS ion mobility–mass spectrometry (IM-MS) and a post-column addition (PCA) iKey Separation Device to:

• Authenticate and screen ginsenoside isomers in complex extracts.
• Measure nitrogen-based traveling-wave collision cross section (TW CCS N2) values for isomer differentiation.
• Assess sensitivity gains and cost reductions by adding organic solvent post-column.

Methodology and Instrumentation

The analytical setup comprised:

• ACQUITY UPLC M-Class System with an ACQUITY UPLC BEH C18 PCA iKey Separation Device (150 µm × 50 mm, 1.7 µm) at 40 °C.
• Microflow rate of 2 µL/min for the separation channel and 1 µL/min of isopropanol post-column addition.
• SYNAPT G2-Si mass spectrometer operating in ESI negative mode (capillary voltage 2.6 kV, lockmass leucine enkephalin, IMS T-Wave ramp from 1000 to 300 m/s).
• Sample set: Korean ginseng tea, red panax and ginkgo biloba + red panax extracts (diluted 1:10), and ginsenoside standards (100 pg/µL).

Key Results and Discussion

Combining UPLC retention time and ion mobility drift time yielded a three-dimensional separation, markedly increasing peak capacity:

• Base peak chromatograms of Korean ginseng extract revealed complex profiles, while drift time vs. retention time plots resolved overlapping features.
• Coeluting isomer pairs Rg1/Re and Rb2/Rc were distinguished by their distinct TW CCS N2 values.
• Drift-time aligned product ion spectra provided clean fragmentation patterns for each isomer (e.g., Rg1 at m/z 845.4897, Re at m/z 991.5484).
• Measured CCS values matched library data within 2 % error: Rb2/Rc (355.24/344.50 Å2), Rd/Re (328.31/323.46 Å2), Rf/Rg1 (301.60/292.03 Å2).

Benefits and Practical Applications

The ionKey/MS IM-MS workflow delivers:

• Enhanced selectivity through an additional separation dimension, resolving coeluting isomers without extended chromatography.
• Improved sensitivity by post-column addition of organic solvent, reducing matrix suppression.
• Reduced reliance on expensive high-purity reference standards, cutting assay costs (e.g., £2483 savings for standards purchase).
• Streamlined data processing and identification using the UNIFI Scientific Information System and a custom TW CCS N2 library.

Future Trends and Potential Applications

Expanding ion mobility workflows could include:

• Building comprehensive CCS libraries for diverse phytochemicals and other bioactive compounds.
• Integrating real-time IM-MS screening in quality control laboratories for regulatory compliance.
• Applying microflow IM-MS to metabolomics, pesticide screening, and authentication of herbal supplements.
• Advancing automation and high-throughput capabilities through improved hardware and software integration.

Conclusion

The combination of microflow UPLC with ionKey/MS ion mobility and PCA post-column addition offers a powerful, cost-effective strategy for unambiguous identification of ginsenoside isomers in functional food products. This approach enhances separation, sensitivity, and confidence in screening workflows, paving the way for broader adoption in quality assurance and research settings.

References

1. McCullagh M., Goscinny S., Hanot V., Tyldesley Worster R., Douce D. Exploring the benefits and potential of iKey microfluidic chromatography and time-of-flight mass spectrometry for pesticide residue analysis. Waters Application Note 720005195en, 2014.
2. McCullagh M., Lewis R., Douce D. UPLC ion mobility mass spectrometry: a new approach to authentication and routine screening of ginsenoside isomers in functional food products. Waters Poster PSTR134803065, ASMS Baltimore, 2014.
3. Ligor T., Ludwiczuk A., Wolski T., Buszewski B. Isolation and determination of ginsenosides in American ginseng leaves and root extracts by LC-MS. Anal Bioanal Chem. 383(7–8):1098–1105, 2005.