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

Importance of the Topic

Precise identification of carbon–carbon double bond locations in unsaturated fatty acids is fundamental to lipidomics, metabolic profiling, and biomarker discovery. Conventional low‐energy collision‐induced dissociation (CID) often only targets polar headgroups and fails to reveal intra‐chain structural details. Novel radical‐driven fragmentation techniques, such as oxygen attachment dissociation (OAD), address this gap by generating diagnostic cleavages at double bond sites without requiring chemical derivatization.

Objectives and Study Overview

This work presents the application of negative‐mode OAD‐MS/MS on the Shimadzu LCMS‐9050 Q‐TOF platform for structural analysis of unsaturated fatty acids. The goals were to integrate OAD into an existing liquid chromatography–tandem mass spectrometry (LC‐MS/MS) workflow, compare its performance against CID and hydrogen abstraction dissociation (HAD), and demonstrate reliable assignment of C=C positions in complex lipid extracts.

Methodology and Instrumentation

Analytical conditions combined reversed‐phase ultra‐high‐performance liquid chromatography (UHPLC) with negative electrospray ionization (ESI) MS/MS. Key parameters included:

• LC system: Shimadzu Nexera X3
• Column: Acquity UPLC Peptide BEH C18 (50 × 2.1 mm, 1.7 μm; Waters)
• Mobile phases: A—ACN/MeOH/water (1:1:3) with 5 mM ammonium acetate and 10 nM EDTA; B—100% IPA with same additives
• Flow rate: 0.3 mL/min; gradient from 0% to 95% B in 20 min
• MS: Shimadzu LCMS‐9050 Q‐TOF configured for Auto‐MS/MS Top 10; negative ESI, collision energy +10 V
• OAD unit: microwave discharge generating neutral O• radicals (<20 W, 2.45 GHz) introduced post‐ionization

Sample preparation followed Uchino et al. (2022), extracting lipids from mouse brain tissue spiked with fatty acid standards.

Key Results and Discussion

OAD‐MS/MS produced characteristic fragment ion pairs bracketing each C=C bond, enabling unambiguous localization of double bond positions in oleic, linoleic, vaccenic, and arachidonic acids. Compared to CID, OAD provided direct bond‐specific information rather than headgroup cleavage. HAD also yielded sequential chain fragmentation but proved less sensitive and more complex in mixtures. LC‐OAD‐MS/MS achieved baseline separation of coeluting isomers and consistent diagnostic signals across retention times.

Benefits and Practical Applications

• No derivatization step is required, reducing sample handling and potential artifacts.
• OAD integrates seamlessly into existing LC‐MS/MS workflows.
• High sensitivity and specificity for double bond assignment supports detailed lipid profiling in biomedical research, quality control, and food analysis.

Future Trends and Potential Applications

Further developments may include high‐throughput OAD‐enabled lipidomics, extension to other classes of unsaturated biomolecules, and coupling with ion mobility for enhanced isomer separation. Integration into routine QA/QC pipelines and real‐time monitoring of oxidative modifications are promising directions.

Conclusion

The implementation of negative‐mode OAD on the Shimadzu LCMS‐9050 Q‐TOF provides a robust, derivatization‐free approach for pinpointing double bond positions in unsaturated fatty acids. Its superior structural specificity over CID and compatibility with UHPLC workflows make it a valuable tool for advanced lipidomic investigations.

References

• Takahashi H., Okamoto M., Miyazaki Y., Arao Y., Asano N. Anal. Chem. 2018, 90 (12), 7230.
• Uchino H. et al. Commun. Chem. 2022, 5, 162.