2.8.4. Drug Phase Partitioning During In Vitro Lipolysis

The solubilization of FEN in the aqueous phase during in vitro gastrointestinal lipolysis was measured. Aliquots (1 mL) were withdrawn at 1, 15, and 30 min (gastric phase) and 1, 5, 10, 15, 30, 45, and 60 min (intestinal phase) and transferred into 1.5 mL centrifuge tubes prefilled with 10 µL of 4-BBA (0.5 M in methanol) as a lipase inhibitor. Samples were centrifuged (32,760 rcf, 10 min, 25 ◦C) to separate the aqueous phase, containing solubilized FEN, and precipitated pellet. The supernatant was appropriately diluted prior to analysis by HPLC. The supernatant was discarded to leave the pellet and was re-dispersed through addition of 1 mL methanol, vortex mixed for 30 s, followed by sonication for 45 min. The tube was centrifuged (32,760 rcf, 10 min, 25 ◦C), and the supernatant was diluted and analyzed by HPLC.
