A slight reduction in the lipid concentration may occur during dispersion production by dilution with process water remaining in the homogenization device. In order to achieve the lipid mixing ratio of 9 CN + 1 TM accurately, the lipid content of the unloaded dispersions was determined by HPLC after preparation. A Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an LPG-3400SD pump, a WPS-3000TSL autosampler, and a Corona Veo Charged Aerosol detector was used to perform the analysis. The column (Thermo Fisher Scientific Hypersil Gold C18, 2.1 × 150 mm, 1.9 μm) was kept at 25 °C and the flow rate was set to 0.3 mL/min. The mobile phase consisted of acetonitrile/tetrahydrofuran 70/30 (v/v). Under these conditions, the retention time of both lipids was between 3 and 5 min, with cholesteryl nonanoate eluting from the column prior to trimyristin.
For sample preparation, dispersions were dissolved in tetrahydrofuran/acetonitrile 50/50 (v/v) and diluted to an appropriate detector response; 1 μL was injected and detected at a nebulizer temperature of 50 °C. Every sample was diluted twice and every dilution measured two times (n = 4). Lipid concentrations were calculated with the Chromeleon 7.2 software (Thermo Fisher Scientific, Waltham, MA, USA) using a calibration curve for trimyristin or cholesteryl nonanoate in different concentrations.
For sample preparation, dispersions were dissolved in tetrahydrofuran/acetonitrile 50/50 (v/v) and diluted to an appropriate detector response; 1 μL was injected and detected at a nebulizer temperature of 50 °C. Every sample was diluted twice and every dilution measured two times (n = 4). Lipid concentrations were calculated with the Chromeleon 7.2 software (Thermo Fisher Scientific, Waltham, MA, USA) using a calibration curve for trimyristin or cholesteryl nonanoate in different concentrations.