Cbm 20a interface

The permeation studies through the hydrogels were performed with a series of beta-blocking agents with a homologous structure and different degree of hydrophilicity—atenolol, betaxolol hydrochloride, penbutolol sulphate, and timolol maleate—using horizontal permeation cells. The cells were composed of two parts, a donor and a receiving compartment. The hydrogels were mounted between the compartments and one side was filled with the drug solution (the donor); meanwhile, the other side contained the Ringer–Krebs buffer (RKB, the receiving phase). The process was carried out at a constant temperature of 32 °C, using a temperature-controlled water bath.
At appropriate time intervals, 1 mL of the receiving solution was withdrawn and replaced with the same volume of fresh RKB. Each experiment was conducted for 4 h and was performed in triplicate.
The withdrawn volume was analyzed by HPLC using a LC-20AT system equipped with an SPD-10A UV detector, a CBM-20A interface (Shimadzu, Kyoto, Japan), and a 20 μL Rheodyne injection valve. The analyses were conducted with a C18 Bondclone (10 μm, 300 × 3.9 mm; Phenomenex, Torrance, CA, USA) column and the conditions are listed in Table 2.
From the obtained data, the apparent permeability coefficient (P_app) values were calculated according to Fick’s first law.

High resolution mass spectral (HRESITOF) analysis was performed on a micrTOF II - ESI-TOF (Brucker Daltonics, Billerica, MA, USA). The capillary voltage was monitored at 4500 V (120 V output). A solution of NA-TFA (10 mg/mL) was used for Internal calibration (TOF). N₂ was used at 200 °C; flow and pressure were set at 4 L/min nebulization and 0.4 bar. Liquid chromatography was performed with a Prominence liquid chromatography system model LC-20A from Shimadzu Technology (Kyoto, Japan), fitted with a diode array detector (DAD), SPDM-20A (Shimadzu, Kyoto, Japan) working in the range of 220–250 nm, a two pump LC-20 AD (Shimadzu, Kyoto, Japan), and a DGU-20A-5 degasser; a CBM-20A interface was used (Shimadzu, Kyoto, Japan). The analysis was performed at room temperature. The optimal chromatographic conditions consisted of an isocratic solvent system containing 25 % of acetronitrile in water, delivered to the C18 Phenomenex Onyx Monolithic 3 μm column (100 mm × 4.6 mm I.D.) at a flow rate of 0.3 mL/min. The eluent was monitored at a 225 nm detection wavelength.

The concentration of OLE in the liposomal formulations after lysis of the vesicles by methanol and in the aqueous solutions was determined by HPLC. The apparatus consisted of a LC-20 AT system with an UV SPD-10A detector and a CBM-20A interface (Shimadzu, Kyoto, Japan). The injection valve was a Rheodyne with a capacity of 20 µL, and a Lichrocart^® C18 (5 µm; 250 × 4.0 mm) column was employed. The mobile phase consisted of a mixture of water:acetonitrile:glacial acetic acid (70:29.9:0.1). The flux was 0.5 mL/min, the detection wavelength was 230 nm, and the retention time under these conditions was 8.0 min. The OLE amount in the samples was determined by comparison with external standard curves obtained by adding increasing amounts of the product to an appropriate solvent. The calibration curves were obtained by applying a least-squares linear regression analysis to experimental data using Prism software, version 8.0 (GraphPad Software Inc., San Diego, CA, USA) and were described by the following equations:a.

y = 27,000x − 1304; R² = 0.9987, at a concentration ranging from 0.425 to 4.000 μg/mL in methanol (Limit Of Quantification = 0.093 μg/mL), to determine the entrapment efficiency;

b.

y = 39,780x + 982; R² = 0.9980, at a concentration ranging from 1.00 to 11.60 μg/mL in water (Limit Of Quantification = 0.322 μg/mL), for stability studies.