Unraveling the Complexity of Lipidomes by Multiple Heart-Cutting Q-TOF LC/MS with the Agilent 1290 Infinity 2D-LC Solution

Significance of the Topic

Comprehensive lipid profiling is critical for understanding biological processes and disease states, as lipids play essential roles in cell structure, signaling and energy storage. However, the structural diversity and dynamic range of lipids in complex biological samples pose analytical challenges. Advanced separation techniques coupled with high-resolution mass spectrometry are needed to improve lipid identification and quantification while reducing interferences.

Objectives and Study Overview

This study demonstrates a fully automated two-dimensional liquid chromatography method with multiple heart-cutting to dissect the lipidome of human blood plasma and induced sputum. The approach integrates hydrophilic interaction chromatography for class-based fractionation and reversed-phase UHPLC for detailed separation of individual lipids, followed by accurate-mass Q-TOF detection. The goal is to reduce coelution of isobaric species and enhance confidence in lipid assignments.

Methodology and Instrumentation

Samples of plasma and sputum underwent MTBE-based lipid extraction with phase separation. The first chromatographic dimension used a custom RX-SIL HILIC column under a gradient of formic acid in acetonitrile and ammonium formate in a mixed solvent, producing five to six polarity-based fractions. Selected fractions were automatically heart-cut and transferred via loops to a ZORBAX Eclipse Plus C18 RRHD column for reversed-phase separation at elevated temperature. Mass spectrometric detection employed Agilent JetStream ESI in both positive and negative modes, collecting centroid data over m/z 200–1700.

Key instrumentation:

• Agilent 1290 Infinity 2D-LC solution with binary pumps, autosampler, column compartments and multi-heart-cutting valve kit
• Agilent 6530 Accurate-Mass Q-TOF LC/MS system with JetStream source

Main Results and Discussion

The HILIC step grouped lipids by head-group polarity, separating neutral lipids (free fatty acids, glycerides, cholesterol esters) from sphingolipids and glycerophospholipids. Orthogonality with reversed-phase UHPLC provided hydrophobicity-based resolution within each fraction. Triglycerides and cholesterol esters were analyzed as ammonia adducts in positive mode. Sphingolipids and glycosphingolipids were preferentially detected in negative mode. Phosphatidylinositols, phosphatidylethanolamines, phosphatidylcholines, sphingomyelins and lysophospholipids were identified in distinct heart-cut windows. A clear benefit was the separation of isobaric phosphatidylcholine and phosphatidylethanolamine species, eliminating confusion in mass spectra and improving quantification.

Benefits and Practical Applications of the Method

• Reduced sample complexity entering the mass spectrometer and lower risk of isobaric interference
• Automated multidimensional workflow suitable for high-throughput lipidomic studies
• Enhanced confidence in lipid identification through class-based fractionation and high-resolution detection
• Applicability to various biological matrices beyond plasma and sputum

Future Trends and Applications

Emerging developments include integration with ion mobility spectrometry for additional separation of structural isomers, extension to targeted quantitative assays for clinical biomarkers, and coupling with high-throughput sample preparation platforms. The workflow may also be adapted for other metabolomic classes requiring complex multidimensional separations.

Conclusion

The Agilent 1290 Infinity 2D-LC solution with multiple heart-cutting effectively unravels complex lipidomes by combining HILIC-based class fractionation and reversed-phase sub-class separation. This automated approach reduces isobaric overlap, enhances lipid identification reliability and supports detailed profiling in plasma and sputum. The method holds promise for advanced lipidomics in research and quality control settings.

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