Around 1 ml of collected cell media or Krebs’-Ringer’s solution was spiked with 10 ng of d4-serotonin (internal standard, IS). Liquid-liquid extraction was performed to purify the sample before LC-MS analysis. A Dionex UPLC pump connected to a Q-Exactive Orbitrap mass spectrometer was used for LC-ESI-MS/MS analysis (Thermo Scientific, CA). A Kinetex 2.6 µm Biphenyl UPLC column (100 mm × 2.1 mm) equipped with a guard column was used for separation. The mobile phases were (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. Multiplexed targeted-MS/MS scan function was used for data acquisition. For each sample, integrated peak area of extracted ion chromatogram of fragment ion m/z 160.075 was normalized to that of m/z 164.100 (fragment ion from IS) for determination of the concentration of serotonin. Standard calibration curves were generated for accurate quantification of serotonin in the cell medium and in the Krebs’-Ringer’s solution.
