Enhancing Analysis Specificity and Deconvolution of Natural Products Using a Positive Mode, Ion Mobility Mass Spectrometry Library

Significance of the Topic

Natural products offer complex matrices with isomeric and coeluting compounds challenging to analyze by liquid chromatography–mass spectrometry alone. Incorporating ion mobility collision cross section (CCS) measurements adds an orthogonal metric to mass and retention time, enhancing specificity and reducing false positives in non-targeted screening.

Objectives and Study Overview

This work aimed to develop a positive-mode ion mobility mass spectrometry library for small-molecule natural products by combining accurate mass, retention time, CCS, and drift-aligned product ions. The library was evaluated using green tea extract analysis, including fortification with known reference analytes, to assess identification confidence and deconvolution performance.

Methodology

The study employed reversed-phase UHPLC and high-resolution ion mobility MS to generate and validate the natural products library. Key method parameters included:

• Chromatography: C18 column, gradient elution with 0.1% formic acid in water and acetonitrile, 45 °C column temperature, 0.75 mL/min flow.
• Mass spectrometry: Positive-ion ESI, m/z 50–1200 acquisition, traveling wave ion mobility, low/high collision energy ramp for HDMSE fragmentation.
• Data processing: Triplicate screening of analytical standards, retention time, exact mass, CCS extraction, and drift-aligned product ions integrated into the spectral library.

Used Instrumentation

• SYNAPT G2-Si ion mobility quadrupole time-of-flight mass spectrometer
• ACQUITY UPLC I-Class system with BEH C18 column (2.1 × 100 mm, 1.7 µm)
• MassLynx v4.1 and UNIFI v1.92 software platforms

Main Results and Discussion

Application to green tea extract demonstrated robust deconvolution of coeluting isomers through ion mobility separation. The library enabled unambiguous identification of flavones, flavanols, alkaloids and glycosides with mass errors <3.2 ppm and CCS variance <1%. Fortification experiments confirmed correct detection of added analytes. CCS fingerprints differentiated closely eluting isomeric pairs such as homoorientin/orientin and isovitexin/vitexin. Alternative LC gradients still yielded confident annotations when screening by CCS and m/z alone, highlighting library flexibility.

Benefits and Practical Applications

Integrating CCS into spectral libraries enhances analytical specificity, reduces false positives, and increases peak capacity for non-targeted profiling of complex botanical extracts. The approach supports rapid screening workflows in phytochemical research, food safety, and natural product discovery.

Future Trends and Potential Applications

Advancements include expanding CCS libraries to broader compound classes, automated conformer and protomer characterization, and coupling ion mobility screening with high-throughput UHPLC platforms. Integration into machine-learning models could further refine identification and quantification workflows.

Conclusion

The developed positive-mode natural products library combining retention time, accurate mass, CCS, and product ions delivers robust specificity and flexibility for complex sample analysis. Ion mobility separation and CCS fingerprinting enable confident identification of isomeric and low-abundance compounds, supporting diverse applications in analytical chemistry.

Reference

1. Goscinny S, McCullagh M, Far F, De Pauw E, Eppe G. Rapid Commun Mass Spectrom. 2019;33(S2):1–15.
2. McCullagh M, Eatough D, Hanot V, Goscinny S. Waters Application Note 720005028EN. 2014.
3. McCullagh M, Giles K, Richardson K, et al. Rapid Commun Mass Spectrom. 2019;33(S2):1–11.
4. McCullagh M, Pereira CAM, Yariwake JH. Phytochemical Analysis. 2019;30(4):1–13.
5. McCullagh M, Douce D, Van Hoeck E, Goscinny S. Anal Chem. 2018;90:4585–4595.
6. McCullagh M, Wood M, Mistry N, et al. ASMS Conference. 2019.
7. Goshawk J, Barknowitz G, McCullagh M. ASMS Conference. 2019.
8. Pringle SD, Giles K, Wildgoose JL, et al. Int J Mass Spectrom. 2014;26:1–12.
9. Giles K, Pringle SD, Worthington KR, et al. Rapid Commun Mass Spectrom. 2004;18:2401.
10. Engelhardt UH, Finger A, Kuhr S. Z Lebensm Unters Forsch. 1993;197(3):239–44.