Liposomal Vitamin D3 Formulation Analysis

The size distribution of liposomes in the liposomal vitamin D₃ formulation was determined by the dynamic light scattering method with some modifications in the preparation of the measured samples due to the presence of pectin (Zetasizer Nano ZS, Malvern, UK). The quantity of vitamin D₃ was determined with RP-HPLC (Reversed-Phase High Liquid Chromatography) according to the method developed by Sazali et al. [39 (link)], with some modifications. The modular HPLC set composed of a pump (Azura P4.1S, KNAUER, Berlin, Germany), autosampler (Marathon Basic, Spark Holland, Emmen, The Netherlands), peltier column thermostat (Jetstram II Plus, Knaure, Berlin, Germany), and a UV-VIS detector (Azura UVD 2.1L, KNAUER, Berlin, Germany) was used. The separation was achieved using: 4.6 × 250 mm; 5 μm particles, 100 Å pore sizes, column (Eurospher 100-5 C18, KNAUER, Berlin, Germany). The freshly prepared mixture of methanol and water (98:2 v/v) as the isocratic mobile phase was pumped at a flow rate of 1 mL/min at 40 °C. The injection volume was 20 μL. Samples for the calibration curve were prepared at the concentration range of 0.6–9 μg/g of vitamin D₃ in ethanol. Samples of liposomal formulations were dissolved in ethanol at the 4/10 (w/w) ratio, mixed, centrifuged (2800 rpm for 10 min.), and filtered through 0.2 μm cellulose membrane before analysis [40 (link)].