A Method of Simultaneous Analysis for 196 Lipid Mediators and Related Compounds Using Triple Quadrupole LC/MS/MS

Significance of Topic

Lipid mediators are bioactive lipid derivatives that regulate inflammation, immunity and vascular homeostasis. Comprehensive profiling of these compounds is critical for understanding disease mechanisms in allergy, thrombosis and metabolic disorders. Advances in triple quadrupole LC/MS/MS sensitivity and speed have enabled simultaneous quantification of large lipid mediator panels, supporting biomarker discovery and physiological research.

Study Objectives and Overview

This work presents the LC/MS/MS Method Package for Lipid Mediators Ver. 3, designed for simultaneous analysis of 196 target lipid mediators and 18 internal standards (214 compounds total). The study aims to optimize multiple reaction monitoring (MRM) parameters, implement confirmatory transitions for reliable identification and introduce a retention time correction tool to improve isomer discrimination.

Methodology and Instrumentation

The method groups target compounds around 18 isotopic internal standards. Mixed standard and sample solutions are analyzed on a Shimadzu LCMS-8060 triple quadrupole mass spectrometer coupled to a Kinetex C8 column (2.1 × 150 mm, 2.6 μm). A gradient from 10 % to 95 % acetonitrile over 25 min at 0.4 mL/min delivers chromatographic separation. A retention time correcting tool adjusts predicted retention times based on measured values from internal standards, reducing assignment errors to under 0.3 seconds.

Used Instrumentation

• Mass spectrometer: Shimadzu LCMS-8060
• Column: Phenomenex Kinetex C8, 2.1 mm × 150 mm, 2.6 μm
• Ionization: ESI (positive/negative)
• Mobile phases: 0.1 % formic acid in water (A), acetonitrile (B)
• Gradient program: 10 % B to 95 % B over 25 min
• Flow rate: 0.4 mL/min; injection volume: 5 μL; column temperature: 40 °C
• Gas settings: nebulizer 2.5 L/min, drying 10 L/min, heating 10 L/min; DL 250 °C, heat block 400 °C, interface 270 °C; CID gas 230 kPa

Main Results and Discussion

Retention time correction using mixed internal standard injections demonstrated high accuracy: the corrected predicted retention time for PGE2 differed by only 0.005 min from the measured value. Human plasma analysis (30 µL sample, solid-phase extraction) identified and quantified 66 lipid mediators in a single batch, with retention time tolerances of ±0.05 min. Saturation was observed only for highly abundant Lyso-PAF.

Benefits and Practical Applications

• High-throughput profiling of up to 196 lipid mediators in biological samples
• Improved isomer resolution through retention time correction
• Quantitative accuracy at sub-nanomolar levels enables biomarker discovery
• Applicable to disease research, pharmacodynamics and quality control in pharma and clinical laboratories

Future Trends and Possibilities

Integration with high-resolution mass spectrometry and automated data processing will further expand lipidome coverage. Combining this method with machine learning–driven peak annotation could accelerate discovery of novel mediators. Adaptation to multiplexed workflows and miniaturized sample formats promises greater throughput for clinical studies and personalized medicine.

Conclusion

The LC/MS/MS Method Package Ver. 3 offers a robust, high-speed platform for simultaneous analysis of a broad range of lipid mediators. Optimized MRM transitions, confirmatory scans and retention time correction ensure reliable identification and quantitation in complex biological matrices.

Reference

Shimadzu Corporation. LC/MS/MS Method Package for Lipid Mediators Ver. 3, Application Note C182, First Edition Feb. 2019.