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).