2.4.1. Sample Preparation—Solid-Phase Extraction of Serum and Plasma Samples

Blood was harvested and centrifuged to isolate the serum, which was stored at −80 ◦C until time of analysis. Fluticasone stock solutions (1 mg/mL) were prepared and stored in DMSO at −80 ◦C. The standard curve and quality controls (QCs) were prepared by spiking fluticasone intermediate solutions prepared in methanol into K2-EDTA pig plasma to final concentrations of 10, 30, 50, 100, 300, 500, 1000, and 3000 pg/mL and 25, 250, and 2500 pg/mL, respectively. It was determined that plasma served as a surrogate matrix for serum for measurement of fluticasone. The quantitative method described here was adapted from a previously published method [53]. In short, 300 µL of 40 mM ZnSO4/10% ammonium hydroxide was added to 300 µL of plasma standard, QC, blank, or serum sample. The mixture was vortexed briefly and centrifuged at 10,000× *g* for 15 min. An HLB µElution plate was conditioned with 100 µL methanol and equilibrated with 2 × 200 µL ddH2O before 550 µL of plasma or serum supernatant was loaded to the appropriate well of the plate. Each well was washed with 2 × 200 µL of 25% methanol and the fluticasone eluted into a polypropylene 96-well plate with 2 × 50 µL of acetonitrile: methanol (1:1). The eluent was diluted with 150 µL ddH2O before analysis by LC-MS/MS.
