Characterization of Unsaturated Fatty Acids in Negative OAD-MS/MS using LCMS-9050

Significance of the Topic

Accurate mapping of double‐bond positions in unsaturated fatty acids is essential for understanding lipid metabolism, biomarker discovery, and nutritional quality assessment. Conventional low‐energy CID‐MS/MS often lacks the ability to pinpoint C=C locations within long hydrocarbon chains, leading to ambiguous structural assignments. Oxygen Attachment Dissociation (OAD)‐MS/MS offers a complementary radical‐based approach, generating specific fragment ions that directly report on C=C positions without chemical derivatization.

Objectives and Study Overview

This study demonstrates the integration of neutral radical‐based fragmentation—particularly negative‐mode OAD‐MS/MS—into the Shimadzu LCMS‐9050 Q‐TOF platform for detailed structural analysis of unsaturated fatty acids. Key goals include:

• Comparing OAD and Hydrogen Abstraction Dissociation (HAD) with conventional CID for lipid structural elucidation.
• Establishing LC and MS parameters for reliable negative‐mode fragmentation.
• Applying the method to complex biological samples (mouse brain lipid extract) spiked with standard fatty acids.

Methodology and Instrumentation

OAD and HAD utilize neutral radicals (O• and H•) generated via microwave‐driven water vapor discharge. These radicals attach to or abstract hydrogen from the analyte ion, inducing sequential bond cleavages along the hydrocarbon chain. Key analytical conditions:

• LC system: Shimadzu Nexera X3 with a Waters Acquity UPLC Peptide BEH C18 column (50×2.1 mm, 1.7 µm).
• Mobile phases: A = ACN/MeOH/water (1:1:3) with 5 mM ammonium acetate and 10 nM EDTA; B = IPA with same additives.
• Gradient: 0–1 min 0% B; ramp to 64% B by 7.5 min; hold and step to 95% B by 20 min; re‐equilibration to 0% B by 25 min.
• Flow rate: 0.3 mL/min. Injection: lipid extract from mouse brain per Uchino et al. (Commun. Chem. 5, 162, 2022).
• MS: Shimadzu LCMS‐9050 Q‐TOF in negative ESI mode with OAD unit; Auto‐MS/MS Top 10 data‐dependent acquisition; collision energy +10 V.

Main Results and Discussion

Comparison of fragmentation modes on model PC (18:1) shows:

• CID‐MS/MS primarily cleaves polar head groups (m/z 184) but fails to localize C=C bonds within the acyl chains.
• HAD‐MS/MS yields sequential carbon‐chain fragmentation but produces complex spectra, limiting sensitivity in mixtures.
• OAD‐MS/MS generates characteristic fragment pairs on either side of each double bond, providing unambiguous positional information.

In a mouse brain lipid extract, negative‐mode OAD spectra enabled resolution of co‐eluting isomeric fatty acids (e.g., linoleic 18:2 n‐6,9; oleic 18:1 n‐9; vaccenic 18:1 n‐7) by the presence of diagnostic ions at expected m/z values. The method required no prior derivatization and maintained high throughput (<25 min run time).

Benefits and Practical Applications

OAD‐MS/MS in negative ion mode delivers:

• Direct C=C bond localization in complex lipid mixtures.
• Enhanced structural confidence for biomarker identification, lipidomics, and quality control.
• A derivatization‐free workflow compatible with routine LC‐QTOF platforms.

Future Trends and Potential Applications

Emerging directions include:

• Integration of OAD with high‐resolution ion mobility for rapid isomer separation.
• Automation of radical generation for broader applicability to polar lipids and peptides.
• Coupling with data‐analysis pipelines for large‐scale lipidomics and clinical biomarker screening.

Conclusion

The adaptation of OAD‐MS/MS to the Shimadzu LCMS‐9050 provides a robust, high‐throughput method for unambiguous localization of double bonds in unsaturated fatty acids. This neutral radical approach complements existing fragmentation tools and enhances structural insights in lipid analysis without derivatization.

Reference

• Takahashi H., et al. Anal. Chem. 90(12), 7230 (2018).
• Uchino H., et al. Commun. Chem. 5, 162 (2022).