Metabolomics: LIPID ISOMER SEPARATION USING HIGH RESOLUTION CYCLIC ION MOBILITY MASS SPECTROMETRY

Importance of the topic

Understanding structural isomers of bioactive lipids such as galactosylceramide, glucosylceramide and gangliosides is essential for accurate lipidomics, biomarker discovery and elucidation of disease mechanisms. High-resolution separation of isomers addresses analytical challenges posed by identical masses and similar chemical properties, enabling confident identification and quantification in complex biological matrices.

Objectives and study overview

This work aimed to demonstrate the ability of a high-resolution cyclic ion mobility mass spectrometer to resolve closely related lipid isomers. Two pairs of isomeric sphingolipids were selected: galactosylceramide versus glucosylceramide (d18:1/18:0) and gangliosides GD1a versus GD1b (d18:1/18:0). The study compared separation performance across multiple passes through the cyclic ion mobility cell to establish optimal conditions for baseline resolution.

Methodology and instrumentation

Samples of each lipid standard were prepared at 1 ng/µL in infusion solvent and introduced at 5 µL/min into an electrospray ionization source. A quadrupole isolated the deprotonated ions of interest (m/z 726.54 for ceramides, m/z 917.49 for gangliosides). Ions were directed into a trap region, cycled through the ion mobility device up to 20 times, then transferred to a time-of-flight mass analyzer. The number of passes controlled the effective ion mobility resolution.

Main results and discussion

• Galactosylceramide and glucosylceramide: One pass yielded no separation (resolution ~65 Ω/ΔΩ). Five and ten passes provided partial separation with valley depths of ~80% and ~15%, respectively. Twenty passes achieved baseline resolution (resolution ~290 Ω/ΔΩ) with distinct arrival times at 210.53 ms and 214.49 ms.
• GD1a and GD1b gangliosides: Multiple passes gradually improved separation. Three passes gave partial separation, while five passes (resolution ~145 Ω/ΔΩ) produced a clear baseline separation of the doubly charged ions with arrival times at 41.22 ms and 42.58 ms.

The cyclic multi-pass approach directly scales ion mobility resolution and reveals minor isomeric impurities, highlighting its utility for stereochemical purity assessment.

Benefits and practical applications of the method

• Adjustable resolution by varying pass number meets diverse analytical demands.
• Enhanced confidence in isomer identification within lipidomics workflows.
• Detection of trace stereoisomer impurities supports quality control and pharmaceutical research.

Future trends and possibilities

Integration of cyclic ion mobility with chromatographic pre-separation could further enhance throughput and selectivity. Continued advances in ion optics and instrumentation may enable sub-millisecond resolution improvements, extending applications to larger biomolecules and complex metabolite mixtures. Automation of pass selection and data analysis will streamline routine deployment in research and clinical laboratories.

Conclusion

This study demonstrates that a cyclic multi-pass ion mobility mass spectrometer can achieve baseline separation of challenging lipid isomers by tuning the number of mobility cycles. The approach offers a powerful, flexible tool for advanced lipidomics and stereochemical analysis.

Reference

1. Isaac G, Olivos H, Plumb RS. Separation of Galactosyl and Glucosylceramide Isomers Using the SELECT SERIES Cyclic IMS. Waters Application Note 720007539, 2022.
2. Isaac G, Olivos H, Plumb RS. Separation of Ganglioside Isomers Using the SELECT SERIES Cyclic IMS. Waters Application Note 720007592, 2022.