Brain Uptake of PF8380 by LC-MS/MS

Brain uptake of PF8380 was studied using LC-MS/MS. Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight. The antagonist was administered by gavage to get an indication about oral bioavailability and uptake efficacy across the gastrointestinal epithelium. At the indicated times mice were transcardially perfused with ice-cold PBS under deep anesthesia, and the brains were dissected and snap frozen in liquid N₂. Brains were homogenized in a BioPulverizer (BioSpec Products, Bartlesville, OK) and tissue homogenates were weighed and extracted using a modified Bligh & Dyer HCl method [32 (link)]. External calibration was performed for PF8380 in a concentration range of 0.1–2 µM, LPA species were quantitated using LPA-C17 as internal standard. Quantitation of LPA and PF8380 was conducted by LC-MS/MS. Chromatographic separation was performed on a Phenomenex Kinetex HILIC column (2.1 × 100 mm, 2.6 µm). Detection was performed on a Thermo Orbitrap Velos Pro (Thermo Fisher Scientific Inc., Waltham, MA, USA) hybrid mass spectrometer, using a HESI II probe in negative ionization mode. Automated identification and quantitation of LPA and PF8380 was performed by lipid data analyzer, as previously reported [64 (link)].