HPLC-DAD Analysis of Curcumin and Hesperetin

Concentrations of curcumin and hesperetin in samples collected during solubility, dissolution, and permeability studies were determined using HPLC with the DAD detector (HPLC-DAD). Shimadzu Nexera (Shimadzu Corp., Kyoto, Japan) equipped with an SCL-40 system controller, a DGU-403 degassing unit, an LC-40B XR solvent delivery module, a SIL-40C autosampler, a CTO-40C column oven, and an SPD-M40 photodiode array detector were employed in this investigation. For the stationary phase, a Dr. Maisch ReproSil-Pur Basic-C18 100 Å column with 5 µm particle size and 250 × 4.60 mm (Dr. Maisch, Ammerbuch-Entringen, Germany) was used. The mobile phase was HPLC-grade methanol:0.1% acetic acid (80:20 v/v). The mobile phase was vacuum filtered through a 0.45 µm nylon filter (Phenomenex, Torrance, CA, USA). The experimental conditions were as follows: a 1.0 mL/min flow rate, a wavelength of 420 nm for curcumin and 288 nm for hesperetin, and a column temperature of 30 °C. The injection volume differed depending on the assay. For the solubility study, it was 2 µL, whereas for the dissolution and permeability assays, it was 10 µL. The duration of the run was 10 min. The retention time was 6.644 min for curcumin and 4.221 min for hesperetin. Method validation parameters (Table S1) and chromatograms (Figure S1) were placed in the Supplementary Materials.