A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations

Area of Science:

• Pharmaceutical Sciences
• Analytical Chemistry
• Materials Science

Background:

• Lipid nanoparticles (LNPs) are vital drug delivery systems, particularly for mRNA vaccines.
• Accurate lipid analysis is essential for LNP formulation and quality control.
• Novel lipids like CSL3 and PolyEtOx offer improved transfection and reduced allergic reactions.

Purpose of the Study:

• Develop and validate a simultaneous HPLC-DAD/ELSD method for quantifying seven major LNP lipids.
• Assess the method's performance for linearity, precision, accuracy, and sensitivity.
• Apply the method to analyze lipid degradation and LNP formulations.

Main Methods:

• High-performance liquid chromatography (HPLC) with diode array detector (DAD) and evaporative light scattering detector (ELSD).
• Separation on a Poroshell C18 column using a water/methanol gradient with 0.1% TFA.
• Method validation according to ICH Q2(R1) & (R2) guidelines.

Main Results:

• Simultaneous quantification of 7 LNP lipids, including ionizable lipids, cholesterol, phospholipids, and PEGylated lipids.
• High validation metrics: R² ≥ 0.997, precision < 5% RSD, recoveries 92.9–108.5%.
• Low detection (0.02–0.04 µg) and quantitation (0.04–0.10 µg) limits achieved.
• Successful analysis of lipid degradation products and liposome formulations.

Conclusions:

• The validated HPLC-DAD/ELSD method provides a fast, selective, and quantitative analysis of LNP lipids.
• This method is crucial for LNP development optimization, chemical characterization, and stability testing.
• It supports the quality control of LNP-based therapeutics, including mRNA vaccines.