Purospher star rp18e column

Qualitative and quantitative analyses of the lavender and lavandin extracts were performed using HPLC-DAD-UV/VIS. Separation of phenolic compounds and coumarins was achieved using the Hitachi Chromaster system (Tokyo, Japan) with a Purospher STAR RP-18e column (5 μm particle size, 250 mm × 4.6 mm, Merck) at 30 °C. The autosampler temperature was set at 20°C, and the sample injection volume was 20 μL. The mobile phase consisted of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) at a flow rate of 1 mL/min. The gradient conditions were as follows: 10–20% B (0–35 min), 20–35% B (35–60 min), 35–10% B (60–60.1 min), 10% B (60.1–70 min). The freeze-dried extracts (40 mg) were dissolved in 2 mL of 50% ethanol (ultrasound-assisted extracts and macerates) or water (decoctions). The chromatograms were recorded at 250, 280 and 330 nm. The peaks were identified by comparing their retention times and UV-Vis spectra with standards.

For LC, a Purospher Star RP18e column (55 × 2 mm, 3 μm, Merck KGaA, Darmstadt, Germany) protected with a C-18 Guard Cartridge column (4 × 2 mm, Phenomenex, Torrance, CA, USA) and two in-line filters (0.5 μm and 0.2 μm, IDEX, Oak Harbor, WA, USA) was used as stationary phase. Column oven temperature was maintained at 25°C. Unless stated otherwise, the samples to be analyzed were dissolved in ammonium formate buffer (10 mM, pH 7.0) and methanol in a ratio of 30:70 (v/v). For recording of the reporter ligand NO711 by LC-MS, an isocratic elution mode with 10 mM ammonium formate buffer at pH 7.0 and acetonitrile at a ratio of 50:50 (v/v) was employed. The injection volume was set to 10 μL at a flow rate of 350 μL/min. For hit identification by LC-MS, a gradient elution at a flow rate of 450 μL/min and 25°C column temperature was performed according to the following conditions: after starting with 10 mM ammonium formate buffer (pH 7.0)/ acetonitrile at a ratio of 60/40 (v/v) the mobile phase was changed to a ratio of 20/80 (v/v) from 0.01 to 0.05 min and held until 3.5 min. Between 3.50 and 3.55 min the ratio was changed back to the starting conditions and held until 9 min. The injection volume was set to 40 μL.

The RVC encapsulation efficiency (%EE) of the suspensions of LMVV 7.4_{in+ sulfate} (or LUV 5.5_in loaded with RVC, only used in the characterization tests) was determined after ultrafiltration-centrifugation (4000 × g for 20 min) in a Millex 10 kDa regenerated cellulose filtration device (Millipore, Bedford, MA, USA). The RVC present in the filtrate was determined by high performance liquid chromatography (HPLC), using a Prostar 410 instrument (Varian, CA, USA), according to a validated method [15 (link)]. Samples were diluted (1:9) in 50 mM Hepes buffer (pH 7.4) and aliquots were injected onto a Purospher^® STAR RP-18E column (octadecylsilane, 150 x 4.6 mm; Merck KGaA, Darmstadt, Germany). The mobile phase was acetonitrile:phosphate buffer (60:40 v/v, pH 8.0), the flow rate was 1.2 mL/min, and the absorbance was measured at 240 nm [15 (link)]. Aliquots of liposomes without RVC were used as controls. The %EE was calculated using Eq 1:
$\%\mathrm{EE}=\frac{{\text{RVC}}_{\text{trapped}}}{{\text{RVC}}_{\text{total}}}.100$
where RVC_trapped refers to the RVC encapsulated in the liposomes, and RVC_total is the initial (total) amount of RVC added to the formulation. RVC_trapped was calculated as the difference between RVC_total and the amount of free RVC, measured by HPLC in the filtered fraction as described above.