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Immunosuppressive Drugs in Blood on Raptor Biphenyl by LC-MS/MS

LC_CF0686
PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct Ion
1.Ascomycin1.4510809.53756.50
2.Tacrolimus1.4710821.61768.51
3.Sirolimus1.6410931.63864.57
4.Everolimus1.6910975.68908.62
5.Cyclosporin A1.83101219.831202.87
6.Cyclosporin D1.891001233.911216.88
ColumnRaptor Biphenyl (cat.# 9309A52)
Dimensions:50 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Guard Column:Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9309A0252)
Temp.:70 °C
Inj. Vol.:5 µL
Mobile Phase
A:0.05% Formic acid, 5 mM ammonium formate in water
B:Methanol
Time (min)Flow (mL/min)%A%B
0.000.54060
2.000.50100
2.010.54060
3.000.54060
Max Pressure:305 bar
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC
NotesSample 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.