Eurospher 100 5 c18

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)].

Chemical reagents and solvents
were purchased from commercial suppliers
(Sigma-Aldrich, BLDpharm, Fluka, Alfa Aesar, and Abcr) and were used
without further purification. Analytical thin-layer chromatography
was performed using Polygram SIL G/UV254 (Macherey-Nagel) plastic-backed
plates (0.25 mm layer thickness) with fluorescent indicator and Merck
TLC Silica gel 60 F254 aluminum-backed plates. The spots were visualized
by UV light (254 nm). Column chromatography was done using silica
gel 60 (0.040–0.063 mm). NMR spectra were from a Bruker Avance
4 Neo spectrometer (¹H: 400 MHz, ¹³C: 101 MHz).
The center of the solvent signal and the TMS signal were used as internal
standards. Deuterated solvents purchased at Eurisotop were used as
solvents. HPLC experiments were performed using a Shimadzu prominence
HPLC with an autosampler SIL-20A HT, column oven CTO-10AS VP, degassers
DGU-20A, detector SPD-M20A, and pumps LC-20 AD with a KNAUER Eurospher
100-5 C18, 250 × 4 mm column. The software used for data processing
was LabSolutions. Mass spectra were from an Orbitrap Elite mass spectrometer
(Thermo Fisher Scientific, Waltham, MA, USA) using direct infusion
and electrospray ionization (ESI). MS data analysis was carried out
with Xcalibur. The purity of all tested compounds was >95% as determined
by HPLC analysis.