Rapid Phospholipid Characterization Using a Novel Intelligent Workflow on a Tribrid Orbitrap Mass Spectrometer

Importance of the Topic

The detailed characterization of phospholipids is critical for understanding cellular membranes, lipid metabolism and disease biomarkers. High-throughput, high-confidence lipid profiling reduces analysis time and improves data quality for research, quality control and clinical applications.

Objectives and Overview of the Study

The study aimed to develop an intelligent, comprehensive LC-MSn workflow on a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer. This single-run approach integrates multiple dissociation techniques (HCD MS2, CID MS2 and CID MS3) to maximize lipid identification and quantitation throughput and confidence.

Methodology and Instrumentation

The workflow employs:

• Thermo Scientific UltiMate 3000 RSLC with an Accucore C18 column (2.1×150 mm, 2.6 µm) at 45 °C and 260 µL/min.
• 28 min gradient from 60:40 ACN/water to 90:10 IPA/ACN, both with 10 mM ammonium formate and 0.1% formic acid.
• Orbitrap Fusion Lumos tribrid mass spectrometer with a brighter ion source and segmented quadrupole.
• Data acquisition strategies: alternated positive and negative HCD MS2 (15 K resolution) in the 0–18 min window, product-dependent high-resolution CID MS2, and neutral-loss-triggered high-resolution CID MS3 (2 s full-scan cycle) in the 18–28 min window.

Main Results and Discussion

The intelligent workflow identified the highest number of lipid species in a single run compared to positive-only and alternating positive/negative strategies. It achieved comparable coverage to separate positive and negative runs while doubling throughput. Base peak chromatograms confirmed high full-scan frequency and sensitivity at reduced injection times. Comprehensive fragment ion information (PC diagnostic ion m/z 184.0733 and TG fatty acid neutral losses) enabled confident molecular-level assignments.

Benefits and Practical Applications

• Single-run acquisition reduces instrument time by 50% versus separate polarity runs.
• Improved sensitivity allows shorter injection and faster cycle times without sacrificing identification depth.
• Enhanced confidence in phosphatidylcholine and triglyceride isomer assignments through multi-stage fragmentation.
• Streamlined workflow benefits lipidomics research, clinical lipid profiling, and QC in pharmaceutical and biotech environments.

Future Trends and Possibilities of Use

Further integration with automated data-processing platforms and AI-driven annotation will expand lipid coverage. The approach can be extended to complex matrices, novel lipid classes and combined with imaging MS to map spatial lipid distributions in tissues.

Conclusion

The intelligent HPLC-MSn workflow on the Orbitrap Fusion Lumos delivers higher throughput and confidence in phospholipid characterization within a single run. Multi-stage fragmentation and advanced acquisition strategies make it a powerful tool for modern lipidomics.

References

• Reiko Kiyonami, David A. Peake, Yasuto Yokoi, Ken Miller. Poster Note 64451: Rapid Phospholipid Characterization Using a Novel Intelligent Workflow on a Tribrid Orbitrap Mass Spectrometer. Thermo Fisher Scientific; 2015.
• Mitsui Knowledge Industry. LipidSearch 4.1 Software. Tokyo, Japan.