Analysis of cationic liposomes by reversed-phase HPLC with evaporative light-scattering detection

Cationic lipid-mediated drug delivery of small pharmaceutical molecules and biological molecules, such as proteins and DNA, has gained increasing popularity for many in vitro and in vivo applications. For this purpose, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is one of the most widely used and efficient cationic lipids. In this work, a simple and rapid reversed-phase HPLC method was developed for the simultaneous determination of cationic lipid DOTAP and neutral co-lipids cholesterol and phosphatidylcholine (DPPC or DSPC) as well as their degradation products in liposome-based drug formulations. Due to the poor UV absorbance of the lipids and their degradation products, an evaporative light-scattering detector (ELSD) was used to monitor the separation. The HPLC separation was achieved using a Phenomenex Luna C18 column at 50 °C by a linear gradient elution with methanol–water mobile phase at a flow rate of 2.0 mL/min. 0.1% (v/v) trifluoroacetic acid (TFA) was added into the mobile phase to enhance the retaining of the cationic lipid DOTAP. This newly developed method enabled direct analysis of liposomes without solvent lipid extraction, and was validated to be linear, precise, accurate, specific and sensitive. The limit of detection (LOD) and limit of quantitation (LOQ) were determined to be 0.15 and 0.30 μg, respectively, for all the four lipids. The method has been successfully employed in a wide range of lipid-based formulation screening, process development and stability testing. Studies of liposome samples under accelerated thermal conditions revealed that the hydrolysis of DOTAP, DPPC and DSPC followed pseudo-first-order kinetics.