Rapid Analysis of Lipid Nanoparticle Components Using BioAccord LC-MS System

Significance of the Topic

The emergence of mRNA vaccines has driven intense interest in lipid nanoparticle systems as delivery vehicles. Controlling the composition, purity, and potential impurities of the four core lipids (ionizable lipid, cholesterol, phospholipid DSPC, and PEGylated lipid) is critical for safety, stability, and efficacy. Rapid and reliable analytical methods are essential for process development and quality control in vaccine and gene therapy production.

Objectives and Study Overview

This work presents the development and validation of a simple, rapid reversed-phase LC-MS method using the BioAccord platform. The goals were to achieve baseline separation of LNP components, define detection limits for each lipid, and demonstrate applicability to complex lipid matrices and impurity screening.

Methodology and Instrumentation

A Waters BioAccord system was configured with:

• ACQUITY UPLC I-Class PLUS
• ACQUITY TUV Detector
• ACQUITY RDa Mass Detector (ESI positive mode, 50–2000 m/z, 10 Hz scan rate)
• ACQUITY Premier CSH C18 Column (2.1 × 100 mm, 1.7 µm) at 55 °C
• Mobile phases: A (ACN/Water/formate buffer with formic acid), B (IPA/ACN/formate buffer with formic acid)
• Gradient elution at 400 µL/min, 5 µL injection, sample at 10 °C
• Data acquisition and processing via UNIFI under waters_connect

Main Results and Discussion

The method achieved clear separation in the order PEGylated lipid, cholesterol, ionizable lipid MC3, and DSPC. Detection limits were established at 5 pg/µL (25 pg on column) for DSPC, MC3, and PEGylated lipid, and 250 pg/µL (1.25 ng on column) for cholesterol. High-resolution mass spectra confirmed identity and charge states, particularly the polydisperse PEG distributions. A custom UNIFI lipid library enabled automated component identification and fragment matching. Binary comparison tools distinguished spiked LNP components in bovine liver extract and highlighted unique impurity peaks. Broad applicability was shown by profiling all major lipid classes in a complex extract.

Benefits and Practical Applications

• Fast, robust analysis suitable for routine QC environments
• Simultaneous determination of target LNP lipids and process-related impurities
• Low detection limits support trace-level monitoring
• Streamlined workflow with integrated data management and reporting

Future Trends and Applications

As LNP formulations evolve, future developments may include integration of ion mobility for isomer separation, expansion of spectral libraries for novel lipid excipients, and higher throughput workflows. Coupling with advanced informatics and machine learning could further automate impurity profiling and accelerate formulation optimization.

Conclusion

The described reversed-phase LC-MS method on the BioAccord system offers a rapid, sensitive, and reproducible approach for comprehensive lipid nanoparticle component analysis. Its robustness and integration with UNIFI support both development and quality control of LNP-based therapeutics.

References

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