2.4.2. Protein Precipitation of Esophagus Tissue Homogenate

Proximal, mid, and distal sections of esophagus tissue were frozen after harvest, as above, and stored at −80 ◦C prior to homogenization. Representative pieces of tissue were excised from each section, weighed, and homogenized in PBS (1:3) using three 30 s cycles at 5500 rpm with six 2.8 mm ceramic beads in a bead mill homogenizer (VWR). Homogenates were stored at −80 ◦C until time of analysis. The standard curve and QCs were prepared by spiking fluticasone propionate intermediate solutions prepared in methanol into esophagus homogenate from untreated animals at 1, 3, 5, 10, 30, 50, 100, 300, 500, 1000, 3000, and 5000 ng/mL and 4, 40, 400, and 4000 ng/mL, respectively. Each 50 µL standard, QC, blank, or sample was extracted with 200 µL of acetonitrile containing 5 ng/mL of d5-fluticasone propionate internal standard. Samples were vortexed for 5 min, centrifuged at 10,000× *g* for 10 min at 4 ◦C. The supernatant containing fluticasone propionate was transferred to a clean tube and evaporated under nitrogen at 40 ◦C. Samples were reconstituted in 500 µL of 25% methanol and analyzed by LC-MS/MS.
