2.3.2. Encapsulation Efficiency and Drug Loading

Encapsulation efficiency (EE%) was calculated as the ratio between the amount of drug content into the MPs and the amount of drug used for their preparation, by means of the following equation:

Drug loading (DL) was expressed as the amount of SSZ in 100 mg of MPs, according to the following equation:

EE and DL were calculated for all the formulations.

SSZ was analyzed by the HPLC method described by Kabir et al. [19], which was adapted to our experimental conditions. For this, an exact amount of MPs (10 mg) was dissolved in DCM (1 mL), and the polymer was precipitated with ethanol (14 mL). This suspension was then centrifuged at 13,000 rpm for 5 min and the supernatant was removed and filtered through 0.45 μm filters.

The HPLC apparatus consisted of an HPLC Perkin Elmer chromatographic system (Waltham, MA, USA), an Emplower CromaNec XP v.1.0.4 software and a Perkin Elmer diode array detector 235C (Waltham, MA, USA). The mobile phase was composed of acetonitrile and phosphate buffer at pH 3.5 (40 mM) (20:80 v/v). Before use the mobile phase was degassed and filtered through 0.45 μm nylon membrane filters. Flow rate was set at 1 mL/min; the injection volume was 20 μL, a Mediterranea Sea C18 column was used (250 mm × 4 mm, 5 µm particle size; Teknokroma^®, Madrid, Spain) and the wavelength was set at 360 nm. All analyses were performed at 25 ± 0.5 °C. The HPLC method was validated to demonstrate the absence of interference between SSZ and PLGA or α-tocopherol. The method was linear within the concentration range of 4.0–10.0 μg/mL, with a limit of detection (LOD) of 0.492 μg/mL and limit of quantification (LOQ) of 1.489 μg/mL.