ANALYSIS OF LIPID SIGNALING CLASS ANALYTES USING A TRAVELLING WAVE CYCLIC ION MOBILITY SEPARATOR

Significance of the topic

The detailed analysis of lipid signaling molecules is critical for understanding their roles in cellular communication, structural organization and disease mechanisms. Because many lipid classes contain isomeric species with identical masses, advanced separation techniques such as ion mobility spectrometry (IMS) are required to resolve cis/trans and positional isomers that cannot be distinguished by chromatography or mass spectrometry alone.

Objectives and Overview

This study evaluates a travelling‐wave cyclic ion mobility (cIM) separator coupled to time‐of‐flight mass spectrometry (Q‐cIM‐oaTOF) to achieve high‐resolution separation and subsequent collision‐induced dissociation (CID) of isomeric lipid classes. The targets include mono‐ and polyunsaturated free fatty acids (FFAs), glycerophosphocholines (PCs), steroids and prostaglandins. Key goals are to determine the IMS resolution requirements for various chain lengths and double‐bond configurations and to demonstrate structural elucidation via post‐IMS CID.

Methodology and Instrumentation

Sample Preparation and Ionization:

• Free fatty acids and PC standards dissolved in chloroform/methanol; diluted in 0.1 % formic acid in MeOH/water.
• Steroid and prostaglandin standards prepared in methanol with 0.1 % formic acid.
• Electrospray ionization in positive or negative mode, with isomeric pairs combined at equal response levels.

Instrumentation:

• SELECT SERIES Cyclic IMS (Q‐cIM‐oaTOF) equipped with a 98 cm RF ion guide comprising over 600 electrodes.
• Travelling‐wave cyclic ion mobility separator operated in multi‐pass mode to vary ion residence time and mobility resolution.
• Post‐IMS CID performed in the transfer region prior to orthogonal acceleration TOF analysis.

Main Results and Discussion

Chain Length and Double‐Bond Position Effects:

• Mono‐unsaturated FFAs exhibited distinct mobility differences between cis and trans isomers; shorter chains required fewer cIM cycles for separation compared to longer chains with partial chain back‐folding.
• Resolution (Ω/ΔΩ) scaled with chain length and bond position; for example, 9Z/E‐hexadecenoic acid (C16:1) required ~20 cycles (Ω/ΔΩ ≈335) to achieve baseline separation.

Polyunsaturated FAs and PCs:

• Species with two or more double bonds produced broader arrival time distributions; full resolution was not achieved for all polyunsaturated isomers at the tested conditions.

Steroid and Prostaglandin Isomers:

• Steroid pairs such as 17‐OHP and 21‐OHP were resolved with Ω/ΔΩ >200 in 6 cIM cycles, enabling distinct CID spectra for each isomer.
• Prostaglandins TxB2 and 6k‐PGF1a were partially separated in 2D cyclic IM‐MS, and their deprotonated ions yielded characteristic fragmentation patterns.

Post‐IMS CID and Structural Elucidation:

• CID spectra acquired after mobility separation provided clear structural information, confirming the identity of cis/trans configurations and positional isomers.

Benefits and Practical Applications

• Improved separation of isomeric lipid signaling molecules supports more accurate lipidomics profiling.
• Integration of cyclic IMS with CID enables detailed structural elucidation of complex mixtures in a single run.
• Enhanced resolving power can be tailored to specific analyte classes, benefiting QA/QC, biomarker discovery and mechanistic studies.

Future Trends and Opportunities

Advances may include coupling cyclic IMS with liquid chromatography for multidimensional separation, development of higher‐throughput IMSn workflows for deep structural analysis, and application to intact lipidomics in clinical and environmental research. Continuous improvements in instrument design and software will further expand the scope of isomer‐specific investigations.

Conclusion

The travelling‐wave cyclic IMS device demonstrated variable resolution capabilities sufficient to separate a range of lipid isomers, from mono‐unsaturated fatty acids to steroids and prostaglandins. Mobility resolution requirements correlated with chain length and degree of unsaturation. Post‐IMS CID facilitated unambiguous structural assignment, highlighting the value of this approach for comprehensive lipid signaling analysis.

References

1. Giles K, Wildgoose JL, Pringle S, et al. In 62nd Annual ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD, June 15–19, 2014.
2. Wojcik R, Webb IK, Deng L, et al. Lipid and glycolipid isomer analyses using ultra-high resolution ion mobility spectrometry separations. Int J Mol Sci. 2017;18(1).
3. Kyle JE, Zhang X, Weitz KK, et al. Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry. Analyst. 2016;141(5):1649-1659.