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

	Peaks	t_R (min)	Conc. (ng/mL)	Precursor Ion	Product Ion
1.	Ascomycin	1.45	10	809.53	756.50
2.	Tacrolimus	1.47	10	821.61	768.51
3.	Sirolimus	1.64	10	931.63	864.57
4.	Everolimus	1.69	10	975.68	908.62
5.	Cyclosporin A	1.83	10	1219.83	1202.87
6.	Cyclosporin D	1.89	100	1233.91	1216.88

Column

Raptor 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

0.05% Formic acid, 5 mM ammonium formate in water

Methanol

Time (min)	Flow (mL/min)	%A	%B
0.00	0.5	40	60
2.00	0.5	0	100
2.01	0.5	40	60
3.00	0.5	40	60

Max Pressure:

305 bar

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	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 3000 rpm and then centrifuged for 10 minutes at 4300 rpm. The supernatant was directly injected (5 μL) for analysis.

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