Page 139 - CRFTApplication Compendium_090613

Quantitative Analysis of Immunosuppressant
Drugs in Whole Blood Using High Throughput
LC-MS/MS
Marta Kozak1, Raimund Ruzicka2, Vani Bodepudi2, Jeff Zonderman3, 1Thermo Fisher Scientific, San Jose, CA, USA,
2Thermo Fisher Scientific, Fremont, CA, USA, 3Thermo Fisher Scientific, Franklin, MA, USA
Introduction
Immunosuppressant drugs inhibit the body’s immune
system and are used in organ transplant patients to
prevent organ rejection. Liquid chromatography-mass
spectrometry (LC-MS/MS) is a widely-accepted technique
for the determination of immunosuppressant drugs in
whole blood by research laboratories.
This application note describes a fast, sensitive, reliable,
and accurate LC-MS/MS quantitative method for use by
research laboratories for the simultaneous analysis of
tacrolimus, sirolimus, everolimus and cyclosporin A in
whole blood.
Experimental Conditions
Sample Preparation
A protein precipitation solution was prepared by mixing
MeOH containing internal standards (Ascomycin and
Cyclosporin D) with ZnSO4 solution. Blood samples were
processed by adding precipitation solution. The mixture
was vortexed and centrifuged. Supernatant was injected
into the LC-MS/MS system.
HPLC
HPLC analysis was performed using Thermo Scientific
Transcend LX-2 advanced multiplexing system. Samples
were injected into a Thermo Scientific Javelin C18 guard
column at 80 °C and analyzed with a 2-minute gradient
method.
Mass Spectrometry
MS analysis was performed using a Thermo Scientific TSQ
Quantum Ultra triple stage quadrupole mass spectrometer
equipped with an atmospheric pressure chemical ionization
(APCI) source in selective reaction monitoring (SRM) data
acquisition mode. Optimized SRM parameters for all of
the analytes and internal standards are shown in Table 1.
Results and Discussion
Figure 1 displays the representative limits of quantitation
(LOQ) chromatograms for tacrolimus, sirolimus, everolimus,
cyclosporin A, and the internal standards. As shown in
Tables 2 and 3, the intra- and inter-day variability were
excellent. For each analyte, intra-day variability was
determined by processing and analyzing 5 replicates of
each QC sample. Inter-day variability was determined
with 5 replicates of each QC sample in 3 different batches.
The method tested negatively for all interferences and
cross-reactivity. No ion suppression or enhancement was
observed.
Conclusion
A fast, sensitive, reliable and accurate method was
developed for the quantification of tacrolimus, sirolimus,
everolimus and cyclosporin A in whole blood by research
laboratories. The use of column multiplexing technology
allows for a 1 min analytical method, which enhances
sample throughput.
Key Words
• TSQ Quantum
Ultra
• Transcend LX-2
System
• Clinical Research
• Multiplexing
Application
Note: 487b
Table 1: Optimized SRM parameters
Parent Fragment Collision Tube Lens
Compound Ion Ion Energy Offset
Tacrolimus 821.4 768.3 18 190
Sirolimus 931.6 864.5 15 190
Everolimus 975.7 908.4 16 190
Ascomycin 809.4 756.4 18 190
Cyclosporin A 1219.9 1202.9 17 190
Cyclosporin D 1234.0 1216.9 17 190
Table 2: Intra-day variability (%RSD)
Analyte QC1 QC2 QC3 QC4 QC5
Tacrolimus 6.8 4.6 4.9 - -
Sirolimus 6.7 6.1 3.9 - -
Everolimus 8.6 5.1 4.5 - -
Cyclosporin A 5.5 4.6 3.4 3.4 3.5
Table 3: Inter-day variability (%RSD)
Analyte QC1 QC2 QC3 QC4 QC5
Tacrolimus 4.2 4.1 1.7 - -
Sirolimus 4.4 7.0 7.5 - -
Everolimus 7.5 2.3 6.8 - -
Cyclosporin A 1.8 2.0 2.4 1.7 4.7
Assay performance summary
Target Analytes Tacrolimus, Sirolimus, Everolimus and Cyclosporin A
Matrix Whole blood
LOQ 1 ng/mL (Tacrolimus, Sirolimus, Everolimus)
10 ng/mL (Cyclosporin A)
Assay Linearity 1-50 ng/mL (Tacrolimus, Sirolimus, Everolimus)
10-2000 ng/mL (Cyclosporin A)
Analysis Time 2.0 min; 1.0 min with column multiplexing
0.8 1.0 1.2
Time (min)
RT:0.86
RT:0.86
RT:0.86
RT:0.87
RT:0.90
RT:0.92
Ascomycin: I.S.
Tacrolimus
Sirolimus
Everolimus
Cyclosporin A
Cyclosporin D: I.S.
Figure 1:
Chromatogram of
lowest calibration
standard
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