Lc 10at vp

Damna was incorporated into polymeric micelles to form Damna-NS via a dialysis procedure [25] (link). PhCS-g-mPEG polymer and Damna (5–60 % w/w of the polymer) were dissolved in 5 mL DMSO and kept out of the light. To allow the drug to incubate with the polymer, the mixture was swirled at room temperature for 24 h. The mixture was subsequently dialyzed against DI water overnight in a dialysis bag (Spectra/Por® 12,000–14,000 MWCO, Spectrum Laboratories, USA). The DI water was changed every day until the amount of DMSO was lower than the limit of detection (0.5 mg/L) [26] . A milky solution was obtained and stored at 4 ºC. HPLC (Shimadzu, Japan) system equipped with a pump (LC-10AT VP), a controller (SCL-10A VP) and a UV detector (SPD-10A VP) at 282 nm was used to evaluate the amount of Damna incorporated into amphiphilic chitosan nanospheres. A synergi 4 µm, Max-RP 80 A 250 × 4.6 mm column (Phenomenex®, USA) was used with a mobile phase of methanol: 0.02 M KH₂PO₄ (70:30) at pH 3 and a flow rate of 1 mL/min. The encapsulation efficiency (EE) of Damna was calculated as Eq. (1). where W_d is the amount of Damna in nanospheres and W_o is the initial amount of Damna.

The analysis of resveratrol was performed by using an LC-10AT VP (Shimadzu Corporation, Kyoto, Japan) equipped with an SLC-10A system controller, an SIL-10AF auto injector and an SPD-10A UV/visible detector. The separation was performed by using an Apollo C18 5μ column (W.R. Grace and Company, U.S.A.).
The LC–MS analysis of R- and S-warfarin followed the method used in a previous study^{20 (link)}. Briefly, the separation was carried out by using a chiral column Astec Chirobiotic V (Sigma-Aldrich Chemical Co., U.S.A.) and detection was performed under negative ionization mode with selected ion monitoring of mass-to-charge ratio (m/z) of 307 for R- and S-warfarin, and 179 for caffeic acid.

The drug release test was performed as previously described with some modifications [23 (link)]. Briefly, 0.75 mL of CKR12-PLGA-MCZ micelles (MCZ concentration = 1.60 mM) (donor solutions) was added into dialysis tubes (MWCO 6–8 kDa, Spectrum Laboratories, Inc., San Francisco, CA, USA), and dialysis was performed against 30 mL of PBS of pH 7.4 (receiver solution) at 37 °C. At fixed time intervals, the samples (0.75 mL) were collected from the receiver solutions for analysis and replaced with an equal volume of fresh PBS (pH 7.4) to maintain sink conditions. For the HPLC analysis, 10 µL of the sample solution was injected into a chromatograph (LC-10AT VP; Shimadzu Corporation) equipped with a UV detector (SPD-10 A VP; Shimadzu Corporation), an integrator (LC solution, Shimadzu), and a reversed-phase column (CAPCELL PAK C 18 UG 120 S5: 150 mm × 4.6 mm i.d.; Shiseido Co., Ltd.). The mobile-phase composition of MCZ was (A) 2.7% (w/v) ammonium hydrogen tetra-n-butyl sulphate and (B) aqueous solution of acetonitrile (65:35, %) at a flow rate of 1.0 mL/min. The measurement time of MCZ emitted after each time interval was 7 min. The UV detection wavelength of MCZ was 225 nm. The mobile-phase composition of MCZ was acetonitrile (2.7%, w/v) and tetra-n-butylammonium hydrogen sulphate (35:65, v/v).

The concentration of oxytetracycline was determined by high-performance liquid chromatography (HPLC, LC-10Atvp, Shimadzu, Japan) with an InertSustain C18 (4.6 mm × 150 mm, 5 μm, filled with octadecylsilane bonded silica gel) column. Chromatographic conditions were as follows: the mobile phase was methanol–purified water−2% triethylamine (V/V/V = 100:100:1), using 10% glacial acetic acid to adjust the pH to 5–6; flow rate: 1.0 mL; column temperature: 25 °C; sample size: 20 μL; wavelength: 280 nm. The intermediate was determined by liquid chromatography using a mass spectrometer (TU-1900, Beijing General instrument Co., Ltd., Beijing, China) with an InertSustain C18 column, and the chromatographic detection conditions were as follows: mobile phase was methanol–deionized water (V/V = 500:500), pH was 5–6 adjusted with formic acid, flow rate was 0.5 mL/min, detection wavelength was set at 280 nm and 355 nm, sample size was 10 μL. The mass spectrometric conditions were as follows: the ion source was an electrospray ionization source (ESI), positive ion detection mode scanning, ion source temperature was 300 °C, mass charge ratio scanning range was 200~2800. In other experiments, the degradation process of oxytetracycline was determined by ultraviolet spectrophotometry (TU-1900, Beijing General Analysis Instrument Co., Ltd., Beijing, China).