2.3. Quantification of Chrysin Encapsulated in Nanoemulsions

The quantity of chrysin in chrysin-NE was measured using high-performance liquid chromatography (HPLC) coupled with a W2690/5 Autosampler, a 2695 Pump, and a 2998 Photodiode Array Detector (Waters, Santa Clara, CA, USA). Chrysin separation was performed by a Symmetry C18 HPLC Column (XSelect HSS C18 3.0 mm × 75 mm, 2.5 µm, Waters, Santa Clara, CA, USA) and an isocratic elution (60% v/v acetonitrile and 40% v/v water containing 1% v/v acetic acid) at a flow rate of 0.2 mL/min. Chrysin was detected by UV absorption at a wavelength of 270 nm [22]. The HPLC chromatograms were analyzed by Empower 2 software (Agilent, Santa Clara, CA, USA).

The quantity of chrysin in chrysin-NE was determined using the calibration curve generated from the peak areas of authentic chrysin (1–25 µg/mL) (Supplementary Figure S1). The HPLC validation parameters (e.g., linear regression equation, correlation coefficient (R²), limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy) are shown in Supplementary Table S1. The encapsulation efficiency of chrysin in chrysin-NE was calculated as follows:

where C_int and C_NE are the concentrations of initial chrysin added in nanoemulsion and chrysin measured in chrysin-NE, respectively.