Quantification of Tofacitinib in Perfusion Buffers

The concentration of tofacitinib in the perfusion buffers was measured via LC-MS/MS. For the calibration curve (concentration range between 0 and 200 nM), the stock solution was diluted in 75 % MeOH and 10 µl of this diluted standard was added to 40 µl of perfusion buffer. Acetonitrile (150 µl) with 1 % formic acid was added to both calibration curve samples and 50 µl perfusion samples. After vortexing and centrifuging, the supernatant was transferred into vial and 1 µl was injected into the LC-MS/MS system that consisted of an Acquity UPLC (Waters, Milford, MA, USA) coupled to a Xevo TQ-S micro (Waters) triple quadrupole mass spectrometer. The tofacitinib was separated using an Acquity UPLC BEH column (Waters, 2.1 x 50 mm, 1.7 µm. The mobile phase consisted of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in MeOH). Separation was achieved at a flow rate of 300 µl/min under the following gradient conditions: 0 min 95 % eluent A, 2.5 min 50 % eluent A, 4 min 0 % eluent A, 5 min 95 % eluent A. The effluent from the UPLC was passed directly into the electrospray ion source. Positive electrospray ionization was achieved using nitrogen as a desolvation gas with ionization voltage at 2000 Volt. The source temperature was set at 500 °C and argon was used as collision gas. The following SRM transitions were used: m/z 313.10 (parent ion) to m/z 149.01 and m/z 97.90 (product ions).