Characterization and quantification of lipid nanoparticle components and their degradants in vivo using an LC-HRAM MS platform

Importance of the Topic

The ability to characterize and quantify lipid nanoparticle (LNP) components and their degradants in vivo is critical for the development of safe and effective drug delivery systems. High-resolution accurate-mass (HRAM) LC-MS/MS methodologies enable detailed profiling of LNP lipid composition, clearance kinetics, and biotransformation pathways while minimizing sample consumption.

Objectives and Study Overview

This study aimed to establish a single-run HRAM LC-MS/MS platform capable of:

• Simultaneous targeted quantification of five key LNP lipids (DOTMA, DMG-PEG 2000, cholesterol, DSPC, DSPE).
  
• Comprehensive identification of unknown lipid metabolites and oxidative degradants in a biological matrix.
  
• Demonstration of method performance across a wide dynamic range with high sensitivity.
  

Methodology and Instrumentation

Sample preparation involved spiking five LNP lipid standards into bovine liver total lipid extract at nine concentration levels ranging from 0.1 ng/mL to 1 µg/mL. Chromatographic separations were performed on a Thermo Scientific Vanquish Horizon UHPLC system using an Accucore C30 column (2.1 × 150 mm, 2.6 µm) at 50 °C with a gradient of acetonitrile/water and isopropanol/acetonitrile mobile phases containing ammonium formate and difluoroacetic acid.

Mass spectrometric analysis was carried out on a Thermo Scientific Orbitrap Exploris 120. Each injection (2 µL) underwent a full MS scan followed by data-dependent MS/MS and targeted MS/MS (tMS/MS) of selected precursor ions for quantification. Data processing and metabolite identification used Compound Discoverer 3.3 and LipidSearch 5.0 software, with calibration and quantification handled in Chromeleon CDS 7.2.10.

Key Results and Discussion

The method achieved low limits of detection and quantification, notably detecting DMG-PEG 2000 at 0.25 pg/µL only by tMS/MS. DOTMA calibration exhibited excellent linearity (R2 = 0.9995) over four orders of magnitude. Mass accuracy for all lipid peaks remained below 3 ppm. MS/MS fragmentation confidently confirmed structural features such as 18:0 fatty side chains on DSPE and endogenous 18:0 Lyso PC in liver extracts with mass errors under 1.5 ppm. Oxidized DOTMA degradants (<0.1% abundance) were identified using predefined MetID workflows and FISh scoring.

Benefits and Practical Applications

This unified LC-MS/MS approach provides:

• Rapid clearance rate monitoring and biodegradation profiling of LNP lipids in vivo.
  
• High sensitivity quantification with minimal biological sample consumption.
  
• Platform versatility for raw material QC, formulation stability, and DMPK studies across LNP products.
  

Future Trends and Potential Applications

Emerging directions include integration of advanced bioinformatics for automated metabolite annotation, expansion to multi-omics analyses, and adaptation of HRAM workflows to novel nanoparticle carriers. Enhanced software pipelines and AI-driven interpretation will accelerate formulation optimization and regulatory compliance.

Conclusion

The Orbitrap Exploris 120 coupled with Vanquish Horizon UHPLC delivers a robust, sensitive, and selective solution for simultaneous quantification and metabolite profiling of LNP lipid components in complex biological matrices. This platform supports key stages of LNP product development, from raw material testing through preclinical DMPK evaluation.

References

1. Thermo Scientific Orbitrap Exploris Series 3.1 instrument control software documentation.
2. Compound Discoverer 3.3 and LipidSearch 5.0 application notes.
3. Previous studies on 18:0 Lyso PC generation via PC hydrolysis.