Journal of Pharmaceutical and Biomedical Analysis 220 (2022) 115011
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Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.journals.elsevier.com/journal-of-pharmaceutical-and-biomedical-analysis
A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids
involved in nanoparticle formulations
Yannick Mousli , Mathilde Brachet , Jeanne Leblond Chain , Ludivine Ferey *
ARNA INSERM U1212, CNRS UMR 5320, University of Bordeaux, Bordeaux F-33076, France
A R T I C L E I N F O A B S T R A C T
Keywords: Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as
Reversed-phase HPLC those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and
Evaporative light scattering detection control the quality of LNPs. The present study is focused on the development and validation of a high-
Lipid analysis
performance liquid chromatography – diode array detector –evaporative light scattering detector (HPLC-DAD/
Lipid nanoparticles
Validation
ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs:
Stability study 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 for
mulations 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 char
acterization, and stability testing of LNP formulations.
1. Introduction as small interferent RNA or mRNA-based vaccines [3,4].
Such complex nanomedicines require specific quality control and
Liposomal formulations have been one of the most studied drug analytical tools to comply with the regulatory guidances. In particular,
delivery systems. They have moved from the simple goal to improve the identification and the quantification of all lipid species have been
bioavailability of poorly water-soluble drugs to multifunctional drug identified as critical quality attributes (CQAs) by the Food and Drug
delivery platforms to enhance therapeutic efficacy by specific targeting Administration (FDA) [5] and the European Medicines Agency (EMA)
or controlled and sustained drug release. Indeed, liposomes offer [6]. In addition, a general effort has been made in the past years to
tunability of composition, flexibility in physico-chemical properties, homogenize and standardize the characterization methods of nano
ease of surface functionalization, biocompatibility, and biodegrad medicines, to improve their translation rate [7]. To contribute to this
ability. Today, 18 pharmaceutical products based on liposomes are endeavor, it is therefore of great importance to develop efficient
approved for clinical use, which makes liposomes the most advanced methods for lipid quantification in nanoparticles to support formulation
nanoparticle technology [1]. Their range of potential applications is of new products or to control the quality and the safety of final products.
constantly being expanded from the delivery of cancer agents, anti High-performance liquid chromatography (HPLC) using reversed-
fungal, pain, vaccines [2], and recently nucleic acid therapeutics, such phase (RP) mode has been used for the separation and the
* Corresponding author.
E-mail address: ludivine.ferey@u-bordeaux.fr (L. Ferey).
https://doi.org/10.1016/j.jpba.2022.115011
Received 24 May 2022; Received in revised form 5 August 2022; Accepted 17 August 2022
Available online 19 August 2022
0731-7085/© 2022 Elsevier B.V. All rights reserved.
