C 18 silica column

The plant Trewia nudiflora L. was collected in Rajshahi, Bangladesh, in May 2006 and a voucher specimen (DACB 34427) was deposited at the Bangladesh National Herbarium. The air-dried powdered stem bark (1.1 kg) was subjected to accelerated solvent extraction using an ASE 100® system (Dionex, UK) successively with n-hexane, ethyl acetate and methanol. Operating conditions comprised of four static cycles (one cycle = 8 min); oven temperature 100 °C, flush volume 60 %, purge time 150 s, pressure 1400–1500 psi. The methanol extract was successively partitioned with n-hexane, ethyl acetate and butanol. The butanol phase was further fractionated by vacuum liquid chromatography using silica gel 60H (VWR International, UK). The fraction eluted with 35 % methanol in ethyle acetate was chromatographed on a C-18 silica column (10 g, Phenomenex, UK) using a Flash Master Personal® system (Biotage, UK). Elution with 100 % water, followed by gradual increases of acetone, yielded compound (2) (46 mg) as a light brown amorphous solid. Characterisation work was performed by a combination of mass spectrometry and ¹H and ¹³C nuclear magnetic resonance spectroscopy experiments, acquired on a ThermoFinnigan LCQ- Orbitrap and a JEOL- 400 Lambda Delta instrument, respectively.

After the preparation of AMP@RT MIL100 and CD-MO@RT MIL100-AMP, supernatants were collected for EE and payload determination. The pellets were re-dispersed in 1 mM PBS for drug release and degradation studies.
To study AMP release from RT MIL100, AMP@RT MIL100 suspensions were centrifuged at 10,000× g for 10 min, and the pellets were re-dispersed in 4 mL 1 mM PBS. The resulting suspensions contained 0.25 mg/mL AMP@RT MIL100. Aliquots of 400 μL were separately distributed into Eppendorf tubes and mildly rotated at 37 °C. At various times (0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 24 h), one sample was taken and centrifuged (10,000× g, 10 min), and the supernatant was evaluated by HPLC. The same strategy was used in the case of coated nanoparticles.
HPLC analysis was carried out with an Agilent system that includes a tunable UV absorbance detector. A C18 Silica column (4.6 × 250 mm, 5 mm; Phenomenex) was employed with an eluant flow of 0.5 mL/min at 25 °C. The mobile phase comprised 88% buffer (0.2 M TEAA) and 12% methanol. AMP was detected at 254 nm with an injection volume of 10 μL. Additionally, the degradation of RT MIL100 was studied in 1 mM PBS according to the release of trimesate.