Rapid Structural Elucidation of Lipids Including C=C Positions Using Dual-Polarity CID/OAD in a Single LC-MS Run

Importance of the topic

Lipids perform essential biological functions, including membrane structure, energy storage, and signaling. Precise mapping of carbon–carbon double bond positions and acyl chain arrangements in lipid species is critical for understanding disease mechanisms and identifying biomarkers. Traditional collision-induced dissociation (CID) often fails to localize double bonds in complex lipid mixtures without chemical derivatization.

Objectives and study overview

This study demonstrates a rapid, single-run approach for comprehensive structural elucidation of phosphatidylcholines (PCs) in mouse liver extract. By integrating dual-polarity switching with simultaneous CID and oxygen attachment dissociation (OAD) in one LC-MS analysis, the method aims to:

• Determine polar head groups and fatty acyl chain composition via CID fragments.
• Assign sn-positions of acyl chains using intensity differences in negative-mode fragments.
• Localize C=C positions through OAD-derived neutral losses.

Methodology and instrumentation

Mouse liver samples were homogenized and lipids extracted using methanol/chloroform/water partitioning. Analytical setup included:

• Shimadzu Nexera X3 high-performance liquid chromatograph.
• Shimadzu LCMS-9050 quadrupole time-of-flight mass spectrometer equipped with OAD RADICAL SOURCE I.
• Chromatographic column: Shim-pack Scepter Claris (100 mm × 2.1 mm, 1.9 µm).
• Mobile phases: 20 mM ammonium formate in water (A) and 1:1 ACN/IPA (B) at 0.3 mL/min.
• Rapid positive/negative polarity switching: CE 25 V in positive mode, CE 35 V in negative mode, scan range m/z 100–1000.

Main results and discussion

Analysis focused on PC 36:4 ions at m/z 782.5695 [M+H]+ and m/z 826.5604 [M+HCOO]–. Key findings:

• Positive-mode spectra confirmed the PC headgroup fragment at m/z 184 and revealed OAD-induced neutral losses specific to each double bond position.
• Negative-mode spectra provided fatty acyl chain fragments, identifying combinations of C18:3, C18:2, C18:1, C20:4, and C16:0 chains.
• Relative intensities in negative-mode allowed assignment of sn-1 and sn-2 positions: saturated chains at sn-1 and unsaturated at sn-2.
• OAD neutral losses corresponded to predicted mass losses for n-3, n-6, and n-9 double bond locations, enabling unambiguous localization of each C=C in co-eluting isomers.
• The PC 36:4 pool was resolved into three structural isomers: PC 18:2/18:2, PC 18:3/18:1, and PC 16:0/20:4.

Practical benefits and applications

This workflow delivers complete structural information on lipid species in a single LC-MS run without chemical derivatization. Benefits include:

• High throughput by combining polarity switching with CID/OAD in one analysis.
• Enhanced confidence in lipid identification through complementary fragment ion sets.
• Applicability to biomarker discovery, quality control in lipidomics, and studies of lipid metabolism in health and disease.

Future trends and opportunities

As lipidomics expands, integrating OAD-based workflows with automated data analysis and machine learning will accelerate untargeted lipid profiling with position-resolved information. The approach can be extended to other lipid classes and coupled with imaging MS for spatially resolved structural lipidomics in tissues and clinical specimens.

Conclusion

The combination of rapid polarity switching and simultaneous CID/OAD on an OAD-TOF system enables detailed structural elucidation of phosphatidylcholines from complex biological extracts in a single LC-MS run. This strategy streamlines lipid isomer analysis and supports advanced lipidomics research.

Reference

1. Uchino H, Tsugawa H, Takahashi Y, Arita M. Computational mass spectrometry accelerates C=C position-resolved untargeted lipidomics using oxygen attachment dissociation. Commun Chem. 2022;5:162.
2. Ekroos K, et al. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J Lipid Res. 2003;44:2181.