4D Analysis of Lipid Nanoparticles (LNP) using Elute-timsTOF Pro 2 with VIP-HESI source

Significance of the Topic

Lipid nanoparticles (LNPs) have become essential delivery vehicles for mRNA vaccines, as demonstrated during the COVID-19 pandemic. Their complex composition requires advanced analytical workflows to characterize component identity, purity and performance. The integration of ultra-high performance liquid chromatography (UHPLC), trapped ion mobility spectrometry (TIMS) and high-resolution mass spectrometry (HRMS) in a 4D-Lipidomics platform addresses the need for multidimensional separation and detailed profiling of LNP formulations in both standard mixtures and biological matrices.

Objectives and Study Overview

This study aimed to develop and demonstrate a rapid 4D analytical method for the simultaneous separation and identification of four critical LNP lipids: a PEGylated lipid (DMG-PEG-2000), a cationic ionizable lipid (DOTAP), cholesterol and a phospholipid (DSPC). Key goals included:

• Establishing retention time, collision cross section (CCS) and mass-to-charge (m/z) benchmarks for each component.
• Comparing conventional ESI ionization with a VIP-HESI source to assess sensitivity gains.
• Evaluating method performance in both neat solutions and spiked plasma extracts (NIST SRM 1950).

Methodology and Used Instrumentation

A working standard mixture of DSPC, DOTAP, DMG-PEG-2000 and cholesterol (1 ppb–100 ppm) was analyzed using a Bruker Elute UHPLC coupled to a timsTOF Pro 2 with VIP-HESI. Key parameters:

• Column: YMC-Triart C18 (100 × 2.1 mm, 1.9 µm).
• Mobile phases: A – 600/390/10 MeCN/Water/1 M formic acid; B – 900/90/10 IPA/MeCN/1 M formic acid; gradient 50 %–99 % B over 13 min.
• Flow rate: 0.4 mL/min; column temp: 55 °C; injection volume: 2 µL.
• MS: timsTOF Pro 2 in positive mode, default 4D-Lipidomics_pos, VIP-HESI source (nebulizer 2 bar, dry gas 8–10 L/min, dry temp 220–230 °C, end plate offset 500 V, capillary 4000 V).

Main Results and Discussion

The four LNP components were baseline separated by UHPLC and further resolved by TIMS. Observed retention times and CCS values:

• DMG-PEG-2000: 2.9 ± 0.2 min, CCS = 816.1 Å2.
• DOTAP: 6.0 ± 0.1 min, CCS = 280.0 Å2.
• Cholesterol (M–H2O+H): 6.3 min, CCS = 205.9 Å2.
• DSPC: 8.6 min, CCS = 300.2 Å2.

VIP-HESI enhanced ion signal intensities by 5–25× relative to standard ESI, improving detection limits. In plasma extracts spiked with LNP components, TIMS added a critical separation dimension to distinguish PEGylated lipids from endogenous co-eluting species, as evidenced by heatmap and extracted ion mobilograms.

Benefits and Practical Applications

The 4D-Lipidomics workflow offers:

• Rapid profiling of LNP composition with high confidence through orthogonal retention time, CCS and accurate mass measurements.
• Enhanced sensitivity for low-abundance components via VIP-HESI ionization.
• Capability to analyze complex biological matrices, facilitating quality control and bioanalytical method development in LNP-based therapeutics.

Future Trends and Potential Applications

Potential developments include:

• Integration of targeted quantification for pharmacokinetic and biodistribution studies.
• Automation and high-throughput screening of LNP libraries to optimize formulation parameters.
• Expansion of CCS libraries for LNP lipids to support regulatory submissions and cross-laboratory data sharing.

Conclusion

The combined UHPLC-TIMS-HRMS platform with VIP-HESI ionization enables comprehensive 4D analysis of LNP components, achieving rapid separation, enhanced sensitivity and multidimensional confidence in compound identification. This approach supports advanced characterization needs in LNP research, formulation development and quality control.

References

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