Oxygen Attachment Dissociation MS/MS for Differentiation between Cis and Trans Fatty Acids

Significance of the topic

Fatty acid double-bond configuration and position profoundly influence membrane fluidity, signaling pathways and metabolic health. Traditional collision-induced dissociation (CID) methods often fail to pinpoint double-bond location or discriminate cis/trans isomers in complex lipid mixtures. Oxygen attachment dissociation (OAD-MS/MS) and hydrogen attachment/abstraction dissociation (HAD-MS/MS) address these gaps by generating diagnostic fragment ions specific to double bonds, enabling rapid and reliable structural lipidomics in research, food analysis and quality control.

Objectives and study overview

This work aimed to develop and validate novel MS/MS approaches for detailed structural analysis of phospholipids, focusing on:

• Precise assignment of carbon–carbon double-bond positions
• Discrimination between cis and trans fatty acid isomers
• Application of techniques to complex lipid mixtures via LC-MS coupling

Model phosphatidylcholines (PCs) with known unsaturation patterns served as standards to evaluate fragmentation behavior and reproducibility.

Methodology and materials

The study combined two radical-based MS/MS strategies in an ion-trap platform:

• HAD-MS/MS: Hydrogen radical (H•) generated from H₂ gas on a heated tungsten capillary initiates acyl-chain fragmentation; diagnostic 12 Da and 14 Da gaps reveal C=C positions.
• OAD-MS/MS: Oxygen radical (O•) produced by microwave discharge of water vapor selectively oxidizes double bonds, yielding stable epoxide adducts ([M + H + O]⁺) and double-bond specific cleavages.
• LC-OAD-MS/MS: Integration of OAD with a 1 s Auto-MS/MS cycle in LC-ESI-MS enabled high-throughput mapping of double bonds in mixtures of eight phospholipid standards.

Instrumentation used

• LC-ESI-MS: Ultra-high-performance liquid chromatography coupled to electrospray ionization ion-trap MS.
• Prototype MALDI-IT-TOF-MS: Matrix-assisted laser desorption/ionization with ion-trap time-of-flight analyzer.
• Radical generation modules: Heated tungsten capillary for H• and microwave discharge unit for O•.

Main results and discussion

HAD-MS/MS produced continuous series of acyl-chain fragments spaced by 14 Da, with characteristic 12 Da gaps marking double-bond sites. OAD-MS/MS yielded non-dissociative [M+H+O]⁺ epoxide ions and specific cleavage patterns adjacent to double bonds. Key findings:

• C=C Position Assignment: Diagnostic ion intensity ratios accurately located double bonds in PCs such as 16:0/20:4 and 18:1(9Z).
• Cis/Trans Discrimination: [M+H+O]⁺ intensity of trans isomers was reproducibly ~2-fold higher than cis counterparts, enabling ratio-based quantitation in mixed samples within a 1 s acquisition window.
• LC-OAD-MS/MS Performance: High-throughput mapping of double-bond positions in multi-component lipid mixtures with clear chromatographic resolution and spectral clarity.

Benefits and practical applications

These radical-driven MS/MS techniques offer:

• Enhanced structural resolution for unsaturated lipids without complex derivatization.
• Rapid throughput compatible with LC workflows for lipid profiling.
• Quantitative discrimination of cis/trans isomers critical to food science, clinical lipidomics and regulatory QC.

Future trends and potential uses

Advancements may include:

• Integration with ion mobility for multidimensional separation of isomeric lipids.
• Automation and miniaturization for routine clinical diagnostics.
• Expansion to other unsaturated biomolecules (e.g., steroids, terpenes) and environmental pollutants.

Conclusion

OAD-MS/MS and HAD-MS/MS represent powerful complementary approaches for pinpointing double-bond location and stereochemistry in phospholipids. Their compatibility with LC-MS platforms and rapid acquisition enables comprehensive lipid structural analysis, opening avenues for improved biomarker discovery and quality control in various industries.

Reference

1. Takahashi H. et al. Anal. Chem. 2016, 88, 3810.
2. Takahashi H. et al. Anal. Chem. 2018, 56, 7230.