2.4.3. LC-MS/MS Analysis

Liquid chromatography for fluticasone propionate was accomplished using a Shimadzu LC-20AD liquid chromatograph with an XBridge BEH C18 (2.5 µm 2.1 × 50 mm) analytical column (Waters) protected with a guard column. The mobile phase consisted of 0.1% ammonium hydroxide (mobile A) and methanol (mobile phase B), and used a gradient of 50% to 95% mobile phase B over 1.5 min. The flow rate was 0.3 mL/min and the total run time was 5 min. Compounds were detected using a Thermo TSQ Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source in the positive ion mode. The spray voltage was held at 4.0 kV and the vaporizer temperature at 300 ◦C. Fluticasone propionate and d<sup>5</sup> fluticasone propionate (internal standard) were detected by multiple-reaction monitoring (MRM) using the transitions 501 ≥ 293 *m/z* and <sup>506</sup> <sup>≥</sup> <sup>293</sup> *m/z*, respectively. Calibration curves were fit using linear regression with 1/X<sup>2</sup> weighting in Xcalibur® v. 2.0 (Thermo Fisher Scientific, Waltham, MA, USA).
