|Sample Preparation Procedure:|
Human whole blood was fortified with 4 immunosuppressive drugs at 10 ng/mL. For quantitation, cyclosporin D was used as the internal standard for cyclosporin A and ascomycin was used as the internal standard for tacrolimus, sirolimus, and everolimus. The blood sample (100 μL) was mixed with 200 μL of precipitation solution (1:4 v/v 0.2 M ZnSO4:methanol) containing 50 ng/mL of cyclosporin D and 5 ng/mL of ascomycin. The mixture was vortexed for 20 seconds at 3,000 rpm and then centrifuged for 10 minutes at 4,300 rpm. The supernatant was directly injected (5 μL) for analysis.