Page 128 - CRFTApplication Compendium_090613

2 Table 2. Source parameters for APCI probe
Source Parameter Value
Sheath gas: 15
Aux gas: 17
Sweep gas: 1
Discharge Current: 4.6 kV
Capillary temperature: 275 ˚C
S-Lens voltage: 75 V
Vaporizer temperature: 300 ˚C
Validation
Validation consisted of analyzing replicates of quality
controls along with a calibration curve on multiple days.
We also analyzed donor samples previously analyzed
using a validated method on a Thermo Scientific™ TSQ
Access MAX™ triple-stage quadrupole mass spectrometer
and compared the results.
Data Analysis
Data was acquired and processed using Thermo Scientific™
TraceFinder™ software. Ascomycin was used as internal
standard for tacrolimus, sirolimus, and everolimus.
Cyclosporine D was used as internal standard for
cyclosporine A. All of the compounds form ammoniated
adducts (Table 3). Extracted ion chromatograms (XIC)
for individual compounds were reconstructed from the
full-scan data with a mass tolerance of 5 ppm. Figure 1
shows representative chromatograms for analytes at their
respective LOQs and internal standards.
Table 3. Exact masses of the ammoniated adducts of the
immunosuppressant drugs and internal standards
Results and Discussion
Linearity
All compounds were linear within the test kit calibrator
ranges of 1–30 ng/mL for tacrolimus, sirolimus, and
everolimus; and 10–1500 ng/mL for cyclosporine A.
Figure 2 shows representative calibration curves for all
compounds. Standards back-calculated to within 6.3%
for tacrolimus, 10.9% for sirolimus, 14.7% for
everolimus, and 7.5% for cyclosporine A.
Figure 2. Representative calibration curves for
immunosuppressant drugs
Compound m/z Compound m/z
Ascomycin 809.5158 Everolimus 975.6152
Tacrolimus 821.5158 Cyclosporine A 1219.8752
Sirolimus 931.5890 Cyclosporine D 1233.8908
Figure 1. Extracted ion chromatograms with 5 ppm mass windows
for analytes tacrolimus, sirolimus, everolimus (each at 1 ng/mL),
and cyclosporine A (10 ng/mL), and internal standards ascomycin
and cyclosporine D
RT: 0.98
AA: 7328
SN64
Everolimus
1 ng/mL
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
RT: 1.06
AA: 2394941
: SN2971
Cyclosporine A
10 ng/mL
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
RT: 0.96
AA: 1796642
SN3403
Ascomycin
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
RT: 1.06
AA: 12945832
SN9240
Cyclosporine D
RT: 0.96
AA: 63351
SN139 :
Tacrolimus
1 ng/mL
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
RT: 0.98
AA: 11506
SN45
Sirolimus
1 ng/mL
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
0
0.5
1
1.5
0 5 10 15 20 25 30 35
Peak Area ResponseConcentration (ng/mL)
Tacrolimus
Y = 3.242e-2X + 1.07e-3; R^2: 0.9982
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30 35
Peak Area ResponseConcentration (ng/mL)
Everolimus
Y = 7.071e-3X - 3.235e-3; R^2: 0.9810
0
0.1
0.2
0.3
0.4
0 5 10 15 20 25 30 35
Peak Area ResponseConcentration (ng/mL)
Sirolimus
Y = 1.144e-2X - 5.203e-3; R^2: 0.9891
0
1
2
3
4
0 250 500 750 1000 1250 1500
Peak Area ResponseConcentration (ng/mL)
Cyclosporine A
Y = 2.221e-3X + 1.668e-1; R^2: 0.9994