HPLC Quantification of NFD Dissolution

Dissolution samples were diluted with HPLC mobile phase (1:1 v/v) consisting of methanol: water: acetonitrile (36:55:9 v/v). HPLC analysis was undertaken using a Varian Prostar 230 Solvent Delivery Module, a Varian Prostar autosampler 410, and a Varian Prostar 310 UV-visible detector (Varian, Palo Alto, CA, USA). Integration of the peaks was performed with a Galaxie Chromatography Data System (Varian, CA, USA). NFD was eluted on a Thermo BDS Hypersil C18 reverse-phase column (200 × 4.6 mm, 5 μm). The mobile phase was filtered by a hydrophilic 0.45 μm filter (Millipore, Millex-LCR, Merck, Madrid, Spain), and pumped at a flow rate of 1 mL/min. The sample injection volume was 50 μL. The column temperature was kept at 25 °C, and the detector was set at 240 nm. The method used was previously validated with a detection limit of 0.12 μg/mL, while the quantification limit was 0.4 μg/mL [17 (link)].