2.3.2. Determination of Entrapment Efficiency (EE%)

The entrapment efficiency (EE%) of PPZ in the nanoparticles was determined indirectly by the ultracentrifugation technique [31]. Briefly, a freshly prepared colloidal nanosuspension was centrifuged at 20,000 rpm for 30 min using a Beckman Coulter® ultracentrifuge (Georgia, United States). During the centrifugation, the temperature was kept constant at 4°C. Then, the settled-down nanoparticles were collected, and the transparent supernatant was analyzed for the determination of the amount of the unentrapped drug using the HPLC technique (n = 3). Twenty microliters of the samples was injected into a Shimadzu® high-performance liquid chromatographic system equipped with an LC-20AD pump and an SPD-20A UV detector that was previously set at 256 nm. A Hector-M® ODS column (250∗4.6∗5) as the stationary phase and a mobile phase that consisted of acetonitrile : ammonium acetate (0.01 M) : methanol (45 : 45 : 10) with a constant flow rate of 1.5 ml/min were used for liquid chromatography (Bogdanovska et al. 2014; Mandal and Ace 1993). According to ICH guidelines, partial method validation was performed in terms of specificity, linearity, accuracy, and repeatability [32].

For the quantification of PPZ, the standard curve was depicted, and the results showed the linearity over the concentration range of 0.78-25 μg/ml with a correlation coefficient of 0.9988. Finally, the entrapment efficiency (EE%) of the prepared nanoparticles was calculated using the following equation: