Promosil c18 column

The encapsulation efficiency (EE%) of liposomes was carried out by the ultrafiltration method [20 (link)]. Briefly, 500 μL of liposomes suspension was taken into a 0.5 mL centrifugal filter tube (100 kDa, Millipore, Billerica, MA, USA) and centrifuged at 5000× g for 15 min. The supernatant was collected, diluted to 5 mL with methanol and detected by HPLC (Hitachi, Shiga, Japan) at 254 nm. The mobile phase containing methanol and water (70:30 v/v) was pumped through a Promosil C₁₈ column (4.6 × 250 mm, 5 μm, Agela Technologies, Tianjin, China) at a constant flow rate of 0.8 mL/min. The column temperature and injection volume were 40 °C and 10 μL, respectively. The regression equation was y = 2415.09 x + 1989.87 in a range of 10.0–200.0 μg/mL with a good linear relationship (R² = 0.9997). The EE% and drug loading (DL%) were calculated by the following equations: $$EE\%=\frac{W_T-W_F}{W_T}\times 100\%,$$
$$DL\%=\frac{W_T-W_F}{W},$$
where W_T is the total mass of HAS in the liposome suspension (mg); W_F is the mass of free HAS in the filtrate (mg); W is the mass of the freeze-dried liposome formulation (mg). All experiments were measured in triplicate.

All reagents and solvents were purchased commercially and used without further purification. Reaction processes were monitored by thin‐layer chromatography (TLC) and visualized under UV light at 254 and 365 nm, or color reagents. Column chromatography was conducted on silica gel (300–400 mesh) using automatic purification apparatus. ¹H NMR and ¹³C NMR spectra were recorded on Bruker AC400 and Bruker AC600NMR spectrometer respectively in Chloroform‐d or DMSO‐d₆ with tetramethylsilane (TMS) as an internal reference. High‐resolution mass spectra were recorded on triple TOF 5600+ MS/MS system (AB Sciex, Concord, Ontario, Canada) in negative or positive ESI mode. The purity of target compounds was determined by high‐performance liquid chromatography with Promosil C18 column from Agela Technologies (4.6 mm × 150 mm, 5 µm particle size). Mobile phase A was double distilled water containing 0.1% trifluoroacetic acid and mobile phase B was methanol containing 0.1% trifluoroacetic acid. Flow rate was 1 mL min⁻¹ using linear gradients as follows: 0–1 min was 40%B, 1–5 min was from 40%B to 95%B, 5–7 min was 95%B, 7–8 min was from 95%B to 40%B, 8–10 min was 40%B. All the biologically tested compounds confirmed at least 95% purity.