Identifying Double-Bond-Positions of Phospholipids in Mouse Liver by Using Simultaneous Positive/Negative Ion Switching Analysis of LCMS-9050 and OAD-MS/MS

Significance of the Topic

The precise location of double bonds in phospholipid molecules is critical for understanding their biological roles and metabolic pathways. Conventional lipidomics approaches often identify lipid classes and fatty acid compositions but fall short of pinpointing double-bond positions. Emerging fragmentation methods such as oxygen attachment dissociation (OAD) combined with advanced mass spectrometry workflows offer new capabilities for detailed structural lipid analysis, aiding research in lipid metabolism, biomarker discovery, and quality control in biomedical and industrial settings.

Objectives and Study Overview

This study aimed to apply simultaneous positive/negative ion switching analysis on a Shimadzu LCMS-9050 coupled with an OAD source to identify double-bond positions in phosphatidylcholine (PC) species extracted from mouse liver. By integrating collision-induced dissociation (CID) for class confirmation with OAD-MS/MS for diagnostic fragment detection, the research sought to characterize PC(36:4) isomers and distinguish fatty acid double-bond arrangements in a non-targeted lipidomics workflow.

Methodology and Instrumentation

Sample Preparation and Extraction:

• Homogenization of 160 mg mouse liver in methanol using a multi-beads shocker.
• Liquid–liquid extraction with methanol, chloroform, and water to isolate lipid fraction.
• Concentration of the extract and reconstitution in appropriate solvent for LC–MS injection.

Chromatographic Conditions:

• Shimadzu Nexera X3 UHPLC system.
• Shim-pack Scepter Claris C18 column (2.1 mm × 100 mm, 1.9 µm).
• Mobile phase A: 20 mM ammonium formate; mobile phase B: acetonitrile/isopropanol (1:1).
• Gradient elution at 0.3 mL/min flow rate.

Mass Spectrometry Conditions:

• Shimadzu LCMS-9050 Q-TOF with simultaneous positive/negative ion switching.
• Positive mode: OAD-MS/MS using OH/O radicals generated by a microwave discharge source in Q2.
• Negative mode: CID-MS/MS to confirm lipid class and fatty acid chain lengths.
• Collision energy: 25 V (positive) / 35 V (negative).

Main Results and Discussion

Chromatographic and MS Profiling:

• Detection of two isomeric peaks at m/z 782.5695 ([M+H]+) and m/z 826.5604 ([M+HCOO]−), both assigned to PC(36:4).
• Data-dependent acquisition captured CID and OAD spectra for each peak (denoted A and B).

CID-Based Structure Elucidation:

• Negative ion CID revealed fragment ions indicative of polar head loss and neutral loss of fatty acid chains, estimating carbon chain lengths and total double bonds.
• Combination analysis suggested peaks A and B correspond to PC species containing 18:2/18:2 and 16:0/20:4 fatty acids, respectively.

OAD-Based Double-Bond Localization:

• Positive ion OAD-MS/MS generated diagnostic fragments specific to each double bond position.
• Peak A (18:2/18:2) showed neutral losses consistent with linoleic acid double bonds at n-6 and n-9 positions.
• Peak B (16:0/20:4) yielded fragments matching arachidonic acid double bonds at n-6, n-9, n-12, and n-15.
• Alpha-linolenic and gamma-linolenic acid isomers were distinguishable by their unique OAD fragment patterns.

Benefits and Practical Applications

By integrating OAD with positive/negative switching on a high-resolution Q-TOF platform, researchers can achieve:

• Comprehensive lipid structural characterization in a single run, including double-bond localization.
• Enhanced confidence in isomer distinction without relying on external derivatization.
• Improved throughput for non-targeted lipidomics studies in biomedical and industrial laboratories.

Future Trends and Possibilities

Advancements in radical-based dissociation techniques and ion-mobility integration may further refine double-bond analysis. Prospective developments include:

• Automation of spectral annotation using machine learning to accelerate data interpretation.
• Extension to other lipid classes (e.g., phosphatidylethanolamine, sphingolipids) for comprehensive lipidome mapping.
• Application to clinical biomarker discovery and nutritional lipid profiling in complex matrices.

Conclusion

This study demonstrated that simultaneous positive/negative ion switching combined with OAD-MS/MS on the Shimadzu LCMS-9050 enables clear assignment of double-bond positions in PC species from mouse liver extracts. The method distinguishes isomeric fatty acids, enhancing structural lipidomics capabilities and providing a robust tool for metabolic and biomarker research.

Reference

1. Takahashi H. et al. Mass Spectrometry. 2019, S0080.
2. Uchino H. et al. Communications Chemistry. 2022, 5, 162.