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

Abstract

Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and control the quality of LNPs. The present study is focused on the development and validation of a high-performance liquid chromatography - diode array detector -evaporative light scattering detector (HPLC-DAD/ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs: ionizable lipids, the neutral co-lipid cholesterol, phospholipids, hydrophilic polymer-lipids for colloidal stability (e.g., a PEGylated lipid). In particular, this study focuses on two innovative synthetic lipids: a switchable cationic lipid (CSL3) which has demonstrated in vitro and in vivo siRNA transfection abilities, and the palmitic acid-grafted-poly(ethyloxazoline)₅₀₀₀ (PolyEtOx), used as an alternative polymer to address allergic reactions attributed to PEGylated lipids. The HPLC separation was achieved on a Poroshell C18 column at 50 °C using a step gradient of a mobile phase composed of water/methanol mixtures with 0.1% (v/v) trifluoroacetic acid (TFA). This method was validated following ICH Q2(R1) & (R2) guidelines in terms of linearity (R² ≥ 0.997), precision (relative standard deviation on peak areas < 5% for intermediate repeatability), accuracy (recoveries between 92.9% and 108.5%), and sensitivity. Indeed, low detection and quantitation limits were determined (between 0.02 and 0.04 µg and between 0.04 and 0.10 µg, respectively). Due to its high selectivity, this method allowed the analysis of lipid degradation products produced through degradation studies in basic, acidic, and oxidative conditions. Moreover, the method was successfully applied to the analysis of several liposome formulations at two key steps of the development process. Consequently, the reported HPLC method offers fast, versatile, selective and quantitative analysis of lipids, essential for development optimization, chemical characterization, and stability testing of LNP formulations.

Keywords: Evaporative light scattering detection; Lipid analysis; Lipid nanoparticles; Reversed-phase HPLC; Stability study; Validation.

Graphical abstract

Fig. 1

Structures of the seven studied lipids.

Fig. 2

Chromatograms of a standard mixture of the 7 lipids of interest detected by A) ELSD, B) UV at 205 nm, C) UV at 293 nm. Column: Poroshell C18 30 * 3 mm, 2.7 µm. Column temperature: 50 °C. Mobile phase: 13.5%/86.5% (v/v) Water/MeOH + 0.1% (v/v) TFA during 1 min and 100% MeOH + 0.1% (v/v) TFA until 10 min. Flow rate: 0.3 mL.min⁻¹. Each lipid in mixture at 100 mg.L⁻¹ in EtOH. Injected volume: 2 µL.

Fig. 3

A) chromatograms of forced degradation of a standard mixture of 7 lipids using 0.1 M NaOH registered between (a) 15 min (blue) and (b) 2 h (black) after NaOH addition. Intermediate chromatograms were registered at 40 min (pink), 1 h (brown), and 1 h 30 (green). PolyEtOx (RT = 3.2 min), DOPE (RT =7.0 min), DSPE-PEG₂₀₀₀ (RT = 7.6 min), and DSPC (RT = 8.0 min) were totally degraded in these conditions. B) chromatogram showing the separation of a standard mixture composed of PolyEtOx, oleic acid, CSL3, and stearic acid using the developed HPLC method.

Fig. 4

Lipid content of LNP formulations before and after dialysis step. Each bar represents the quantity of lipid compared to the quantity initially introduced in the formulation. LNP01: CSL3/Chol/DSPE-PEG₂₀₀₀/DSPC (50.0/37.5/2.5/10.0, mol%); LNP02: DODMA/Chol/DSPE-PEG₂₀₀₀/DSPC (50.0/37.5/2.5/10.0, mol%); LNP03: CSL3/Chol/PolyEtOx/DSPC (60.0/30.0/2.0/8.0, mol%); LNP04: CSL3/Chol/DSPE-PEG₂₀₀₀/DOPE (60.0/30.0/2.0/8.0, mol%). Each LNP formulation was diluted and analyzed 3 times.