High Sensitivity, High Throughput LC-MS Analysis of Eicosanoids Using the Xevo™ MRT QTof

Importance of the topic

Eicosanoids such as prostaglandins and leukotrienes are key lipid mediators in inflammation and physiological regulation. Their low abundance and structural similarity present analytical challenges, making sensitive and selective quantification essential for research in pharmacology, biomarker discovery, and disease mechanism studies.

Objectives and Study Overview

This work demonstrates a high-sensitivity, high-throughput liquid chromatography–mass spectrometry workflow for quantifying major eicosanoid species. By coupling UltraPerformance LC with the Xevo™ MRT QTof, the study seeks to achieve detection limits and dynamic ranges on par with tandem quadrupole methods, while leveraging the advantages of high mass accuracy and resolution.

Methodology and Instrumentation

Samples containing six eicosanoids were prepared across a 14-point calibration range (0.05–1000 ng/mL) in methanol. Chromatographic separation employed a CORTECS T3 column (2.1×50 mm, 1.6 μm) at 35 °C, with a gradient of 0.01% formic acid in water and acetonitrile. The Xevo MRT QTof operated in MSe mode with negative electrospray ionization, a mass range of 50–1200 Da, and lockspray calibration every 1.5 min to ensure mass accuracy.

Instrumentation Used

• ACQUITY Premier UPLC system with Flow-Through Needle injector
• CORTECS™ T3 Column, 120 Å, 1.6 μm, 2.1 × 50 mm
• Xevo™ MRT QTof mass spectrometer
• Data processing software: waters_connect™, Lipostar 2, Skyline

Main Results and Discussion

The workflow provided limits of detection below 0.05 ng/mL for Leukotriene B4 and 0.1 ng/mL for PGD2, covering four orders of magnitude with linear calibration curves. Triplicate injections at low, mid, and high concentrations exhibited peak area reproducibility with coefficients of variation under 3.5%. Mass resolution exceeded 60 000 FWHM and mass errors were typically below 1 ppm, enabling confident empirical formula determination and fragment identification. The data are compatible with third-party lipidomics tools, offering flexibility in informatics workflows.

Benefits and Practical Applications

This method offers:

• Sub-pg sensitivity and wide dynamic range for low-abundance analytes
• High mass accuracy and resolution for selectivity in complex matrices
• Fast acquisition rates supporting high sample throughput
• Seamless integration with lipidomics software platforms

It is particularly suited for metabolomics and lipidomics research, biomarker quantification, and pharmacodynamic monitoring.

Future Trends and Potential Applications

Future developments may include automated sample preparation, integration with multi-omics approaches, expansion to broader oxylipin panels, and real-time data quality control. High-resolution MS workflows will continue to enable large-scale cohort studies and clinical biomarker validation.

Conclusion

The combination of UPLC and Xevo MRT QTof delivers a robust, high-throughput platform for comprehensive eicosanoid analysis, matching tandem quadrupole sensitivity while providing superior mass accuracy and resolution. This workflow supports reliable quantification and confident identification of lipid mediators across diverse research applications.

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