Determination of tacrolimus intracellular concentrations by liquid-mass spectrometry
The collected cells were resuspended in 100 μL of PBS or medium and mixed with 50 μL of internal standard (ascomycin, 800 ng/ mL, dissolved in methanol), 50 μL of methanol, and 500 μL of methyl-tert-butyl ether. After vortexing and centrifugation (14000×g , 5 min, 4 °C), 450 μL of the organic layer was dried on a nitrogen blow-dry apparatus. The extract was redissolved in 50 μL of a complex solution (2 mM ammonium acetate and 0.1% formic acid). After vortexing and centrifugation (12000×g , 10 min, 4 °C), the supernatant (45 μL) was placed in a liquid injection flask. The mobile phase for UHPLC-MS/MS detection was [acetonitrile (containing 0.1% formic acid): 2 mmol/L ammonium acetate (containing 0.1% formic acid) = 9:1], and the column temperature was 55 °C. The specific product ions were m/z 821.5 and m/z 809.5 for tacrolimus and the internal standard, respectively. Ionisation was carried out in positive ion mode with a capillary voltage of 3.5 kV, a cone voltage of 22/29 (tacrolimus/internal standard), an ion source temperature of 120 °C, a desolvation temperature of 350 °C, a nitrogen flow rate of 600 L/h, a collision pressure of 5×10-3 bar, and a collision energy of 17/21 (tacrolimus/internal standard).