4.5.1. HPLC Assay

Compound assay was performed by HPLC (Merck Hitachi Elite LaChrom apparatus, Darmstadt, Germany), equipped with a L-2400 UV–Vis detector and an L-2130 isocratic pump. The mobile phase A was water, and the mobile phase B was methanol. Gradient steps were programmed as follows: A–B 60%–40% at time 0, ramped to 70% B in 3 min, held at 70% for 2 min and returned to initial conditions during 3 min. A Hibar Purospher^® RP-8e (150 × 4.6 mm, 5 µm pore size) was the stationary phase. UV detection was performed at 292 nm. The injection volume was 20 µL, the flow rate was 1.2 mL/min and the column temperature was 40 ± 1 °C. Under these conditions, the compounds retention time was 5.30 ± 0.01 min. The method was validated for linearity (r² = 0.998; r² = 0.997), limit of quantification (3.03 µg/mL; 4.50 µg/mL) and limit of detection (0.91 µg/mL; 1.35 µg/mL) for P293BL and P297, respectively.