2.8. LC-MS analysis of sildenafil

The concentration of sildenafil in placental perfusate was measured with a FDA validated liquid chromatography-mass spectrometry method (LC-MS), using Thermo TQS Vantage LC-MS/MS for the analysis. Column 2·1 × 100mm Waters Acquity CSH C18 1·7um. The mobile phase A consisted of 2 mM ammonium acetate in 0·1% formic acid in water. The mobile phase B consisted of 2 mM ammonium acetate in 0·1% formic acid in LC-MS methanol. Flow rate was 0·5 mL/min. The mobile phase composition changed linearly during analysis in a percentage mobile phase A (from 80% to 0) and B (from 2% to 100%). Total analysis time was four min. The injected volume was 10 μL. Internal standard was vardenafil. The method was validated according to FDA guidelines between 2 and 1000 μg/L for sildenafil and 2–500 μg/L for desmethylsildenafil [37].