Rapid Quantification and Validation of Lipid Concentrations within Liposomes

Abstract

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and d-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Translating the fabrication of protein-loaded poly(lactic-co-glycolic acid) nanoparticles from bench to scale-independent production using microfluidics

“…The high PLA ratio may be related to higher hydrophobicity and polymer precipitation within the cartridge [2]. Despite the lower polymer recovered observed compared to the high (˜100%) lipid recovery obtained after liposome manufacture using microfluidics showed previously [30], the values are inline with those reported in the literature. Values between 40 and 80% are commonly reported when other techniques such as the double emulsion method (w/o/w), spray-drying or nanoprecipitation have been applied for the manufacture of polymers [31,32].…”

Development and validation of a novel UPLC-ELSD method for the assessment of lipid composition of nanomedicine formulation

“…From an analytical point of view, the quality of these pharmaceuticals must be assured through the development of appropriate methods to characterize and quantify their lipid components and degradation products [18,19]. For this purpose, high-performance liquid chromatography (HPLC) is the technique of choice because of its ability to separate lipids into different classes or species based on their alkyl chain lengths and/or head group polarity [20][21][22][23]. For instance, reversed-phase (RP)-HPLC has become the most popular method for separating and analyzing triglyceride species in oils and fats, because it operates on the principle of both chain length and degree of unsaturation of fatty acids, thus enabling separation of individual triglyceride molecules [24,25].…”

Preparation of DPPC liposomes using probe-tip sonication: investigating intrinsic factors affecting temperature phase transitions

“…Sample overheating can lead to lipid degradation which can affect the final properties of the liposomes, so it was important to carefully monitor samples for increases in temperature while extending the sonication time. 29 Conversely, samples could not be chilled as a measure to prevent overheating during sonication else we introduce the risk of creating conditions whereby DPPC preferentially assumes a solid crystalline-like state. This can hinder liposome formation.…”