Achieve broad lipid quantitation using a high-throughput targeted lipidomics method

Importance of this topic

Lipid molecules play crucial roles in cellular structure and signaling and are key biomarkers in health and disease. High throughput and reliable quantitation of diverse lipid species is essential for research, clinical studies and industrial applications. Combining chromatographic separation with targeted mass spectrometry overcomes limitations of direct infusion methods such as ion suppression and isobaric overlaps.

Objectives and Study Overview

The aim of this work is to present a robust LC based targeted lipidomics assay for the quantitation of approximately 1900 lipid molecular species across major classes and subclasses. The method utilizes time scheduled multiple reaction monitoring on high sensitivity triple quadrupole and QTRAP systems from SCIEX. It is designed for broad coverage, high specificity and throughput, and supports customization for class specific or expanded studies.

Methodology and Instrumentation

• Chromatographic separation based on lipid class to reduce interclass isobaric interference and establish narrow retention windows
• Targeted MRM acquisition with Scheduled MRM Algorithm to maximize sensitivity, specificity and dwell times
• Time scheduled MRM methods built using initial unscheduled runs and optimized with an Excel based sMRM Pro Builder tool
• Dual polarity acquisition to enable molecular species level identification of phospholipids in negative mode and broad coverage in positive mode
• Internal standards from Avanti SPLASH and SCIEX Lipidyzer kits used for retention time determination and relative or absolute quantitation

Instrumentation details:

• ExionLC system with binary high pressure mixing pump, degasser, thermostated autosampler and column oven
• SCIEX QTRAP 6500 Plus with IonDrive Turbo V source or SCIEX Triple Quad 7500 with OptiFlow Pro source
• Data acquisition and method development with Analyst Software or SCIEX OS Software
• Data processing using SCIEX OS with AutoPeak algorithm and sMRM Pro Builder templates

Key Results and Discussion

• Chromatographic reproducibility demonstrated by retention time standard deviations below 0.05 minutes for most lipids across ten plasma injections
• Quantitative reproducibility showed that 80 percent of lipid species in key classes had peak area CVs below 20 percent in replicate analyses
• Negative mode MRM transitions provided molecular species resolution of phospholipids that is not possible using head group loss alone in positive mode
• Scheduled MRM Pro Algorithm and concurrency tools optimized dwell times and cycle times for large MRM panels of nearly 2000 transitions
• Customization of the target list allowed filtering to measurable species, improving throughput and reproducibility for large classes such as triacylglycerols

Benefits and Practical Applications

• Comprehensive lipid coverage including phospholipids, sphingolipids, glycerolipids, sterols and free fatty acids
• Reduced isobaric interference through class based chromatography and time scheduled MRM
• High throughput analysis with 17 minute run times and fast polarity switching capable of less than 5 millisecond transitions
• Wide dynamic range and high sensitivity at short dwell times for reliable quantitation in complex matrices
• Flexible workflow that supports addition of new lipid classes and standards for targeted studies
• Automated data processing with pre built methods to streamline analysis and reporting

Future Trends and Potential Applications

Advances in labeled internal standard mixtures and quantitative strategies will enable more accurate absolute quantitation. Integration with ion mobility separation and high resolution mass spectrometry may further enhance lipid identification and isomer resolution. Automation of method development and data analysis will support larger scale studies, single cell lipidomics and clinical lipid biomarker discovery. Expanding regulatory compliant workflows could drive adoption in diagnostics and precision medicine.

Conclusion

The described LC MRM assay offers a robust platform for quantitative lipidomics with broad species coverage, high reproducibility and adaptable workflows. It leverages chromatographic separation, time scheduled targeted acquisition, advanced software tools and internal standards to deliver reliable results in research and applied settings. The customization options and high throughput make it suitable for diverse lipidomics applications from basic research to clinical studies.

References

1. Han X Yang K Gross RW Multi Dimensional Mass Spectrometry Based Shotgun Lipidomics and Novel Strategies for Lipidomic Analyses Mass Spectrometry Reviews 2012 31(1) 134 178
2. Bielow C Mastrobuoni G Orioli M Kempa S On Mass Ambiguities in High Resolution Shotgun Lipidomics Analytical Chemistry 2017 89 2986 2994
3. Ovčacíková M Lísa M Cífková E Holčapek M Retention behavior of lipids in reversed phase ultrahigh performance liquid chromatography electrospray ionization mass spectrometry Journal of Chromatography A 2016 1450 76 85
4. Koelmel JP Kroeger NM Gill EL Ulmer CZ Bowden JA Patterson RE Yost RA Garrett TJ Expanding Lipidome Coverage Using LC MS MS Data Dependent Acquisition with Automated Exclusion List Generation Journal of the American Society for Mass Spectrometry 2017 28(5) 908 917
5. Hines KM Herron J Xu L Assessment of Altered Lipid Homeostasis by HILIC Ion Mobility Mass Spectrometry Based Lipidomics Journal of Lipid Research 2017 58(4) 809 819