Sensitive cationic lipids impurities analysis with quantitation by charged aerosol detection and simultaneous mass confirmation by MS

Significance of the Topic

Cationic lipids are fundamental building blocks of lipid nanoparticle systems used in the delivery of mRNA, siRNA and other nucleic acids. Their amphiphilic structure enables efficient encapsulation, protection and cellular uptake of genetic materials, making impurity profiling crucial for therapeutic safety and efficacy.

Objectives and Study Overview

This study aimed to illustrate the performance of an inverse gradient LC method on a Vanquish Flex system equipped with a Charged Aerosol Detector HP (CAD HP) and an ISQ EM Single Quadrupole MS. The focus was on high-sensitivity quantitation and repeatable mass confirmation of impurities in three cationic lipid standards: R-DOTAP, DLin-KC2-DMA and ALC-0315.

Methodology

The workflow consisted of:

• Sample preparation: Lipid standards dissolved in methanol or ethanol, vortexed and adjusted to ~1 mg/mL.
• Chromatography: Hypersil GOLD C8 column (3×100 mm, 3 µm) using 5 mM ammonium formate in water/acetonitrile (Solvent A) and methanol (Solvent B). An inverse gradient was applied to enhance detector uptime and sensitivity.
• Detection: CAD HP settings included a 35 °C evaporation temperature, 10 Hz data rate and a diverter valve to waste non-target components. Mass confirmation was performed in positive HESI mode over m/z 300–900.
• Data analysis: Thermo Scientific Chromeleon CDS facilitated acquisition and quantitative evaluation of peak areas, heights and retention times.

Used Instrumentation

• Thermo Scientific Vanquish Flex Inverse Gradient UHPLC system
• Thermo Scientific Vanquish Charged Aerosol Detector HP
• Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer
• Hypersil GOLD C8 HPLC column (3.0×100 mm, 3 µm)

Main Results and Discussion

The CAD HP demonstrated sensitivity sufficient to detect impurity III at its LOQ (S/N > 10) with a calculated concentration of 0.023 mg/mL. Six replicate injections yielded RSD values for peak area and height below 2%, and retention time RSDs below 0.05%. The ISQ EM MS channel confirmed lipid and impurity masses with RSDs of 1–2% for peak areas and <0.15% for retention time. The diverter valve approach improved detector availability and minimized matrix effects.

Practical Applications and Benefits of the Method

• High-throughput impurity profiling for raw material QC in pharmaceutical and biopharma development
• Compliance-ready quantitation with robust repeatability and sensitivity
• Integrated mass confirmation reduces false positives and supports regulatory documentation

Future Trends and Potential Applications

Advances in lipid nanoparticle therapeutics will drive demand for more sensitive detectors and high-resolution mass spectrometry. Automation of inverse gradient workflows, expansion to diverse lipid chemistries and alignment with evolving regulatory guidelines will further strengthen impurity control strategies.

Conclusion

The integrated Vanquish Flex inverse gradient LC-CAD HP and ISQ EM MS setup provides a powerful, compliance-ready solution for quantitative impurity analysis of cationic lipids. It combines high sensitivity, excellent repeatability and straightforward mass confirmation, supporting rigorous quality assurance in lipid nanoparticle research and production.

References

1. Quantifying impurities in cationic lipids raw materials with the inverse gradient method using LC-CAD-MS - Application note 003384
2. Albertsen C et al. The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv Drug Deliv Rev. 2022 Jul 3;188:114416
3. Birdsall R et al. Monitoring stability-indicating impurities and aldehyde content in lipid nanoparticle raw material and formulated drugs. J Chromatogr B. 1234 (2024) 124005