TECHNICAL NOTE 64969

Testing robustness: Immunosuppressant drugs in

blood with a TSQ Quantis MS for clinical research

Authors Goal
Kristine Van Natta, Neloni Wijeratne, Demonstrate the robustness of the Thermo Scientific™ TSQ Quantis™ triple
Claudia Martins quadrupole mass spectrometer by analysis of immunosuppressant drugs in
whole blood for clinical research.
Thermo Fisher Scientific,
San Jose, CA Introduction
Mass spectrometry is a popular choice of technology in clinical research for
the quantitation of immunosuppressant drugs in whole blood. A sensitive and
Keywords robust instrument is required for reliable quantitation workflows. In this study,
Immunosuppressant, we demonstrate the TSQ Quantis triple quadrupole mass spectrometer for
tacrolimus, sirolimus, everolimus, this application.
cyclosporin A, TSQ Quantis MS,
TraceFinder, clinical research, Experimental
robustness Sample preparation
Calibrators and controls were obtained from RECIPE Chemicals +
Instruments GmbH (Munich, Germany). Blank whole blood was obtained from
BioreclamationIVT (New York, USA). Briefly, whole blood calibrators, controls,
and 10 different lots of blank whole blood were processed by precipitation
with ZnSO4/methanol solution containing internal standards (cyclosporin D
and ascomycin). Samples were vortexed for one minute, left to stand for
30 minutes in a refrigerator, and centrifuged at 13,000 rpm for 10 minutes.
Supernatant was transferred to an autosampler vial, and 15 µL was injected
onto the HPLC system. This processing method removes the majority of
blood proteins but does not remove phospholipids.
 Liquid chromatography A
Chromatographic separation was performed using a
Thermo Scientific™ Vanquish™ Flex Binary HPLC system.
The column used was a Thermo Scientific™ Hypersil
GOLD™ C8 LC column (50 × 2.1 mm, 5 µm particle size)
maintained at 80 °C. Mobile phases A and B consisted
of 10 mM ammonium formate with 0.1% formic acid in
Fisher Chemical™ Optima™ grade water and methanol,
respectively. No divert valve was used. The total run time
was 3 minutes.

Mass spectrometry
Compounds were detected on a TSQ Quantis triple
quadrupole mass spectrometer equipped with a heated B
electrospray ionization source. The ion source was set
to Medium (M) at position 2, with sweep gas set to 2
(arb units). All the compounds formed an ammoniated
adduct that was used as the precursor ion. Two selected
reaction monitoring (SRM) transitions were monitored
for each analyte and one was monitored for the internal
standards. Dioctyl phthalate and three endogenous
phospholipids were also monitored as surrogates for
instrument cleanliness.

Test of robustness
The daily sequence of samples consisted of an initial set
of eight calibrators. This was followed by repeated sets C
of five controls and 20 blank blood samples. A set of
calibrators was inserted approximately halfway through
the total sequence and again at the end. The total
number of samples per sequence was approximately
300. The sequence was repeated for five consecutive
days. Peak areas of the internal standards and calculated
concentration of the mid-level control were monitored for
stability.

Data analysis
Data was acquired and processed using Thermo
Scientific™ TraceFinder™ software. Figure 1 shows
representative chromatograms for analytes at their Figure 1A, 1B, and 1C. Chromatograms for lowest calibration
respective LOQs and internal standards. standard showing ion ratio confirmation.

2
D A

B
E F

Figure 1D, 1E, and 1F. Chromatograms for lowest calibration C

standard showing ion ratio confirmation along with internal
standards.

Results and discussion

Linearity
All compounds were linear over their calibration
ranges of approximately 2–60 ng/mL for tacrolimus,
sirolimus, and everolimus, and 25–1800 ng/mL for
cyclosporin A. Figure 2 shows representative calibration
curves for all compounds. All calibrators back-calculated
to within 20% of theoretical values over the five days of
testing.
D

Figure 2. Representative calibration curves.

3
 Robustness 300 A
Peak areas for cyclosporin D and ascomycin, the two 250
internal standards used in this study, showed precisions
200
of 4% and 8%, respectively, over the five days and over

Calculated ng/mL
1500 injections (Figure 3). Calculated concentration 150
Day 1 Day 2 Day 3 Day 4 Day 5
precisions for Control III for cyclosporin A, everolimus, 100
sirolimus, and tacrolimus were 2.36%, 4.22%, 4.08%, and
50
3.57%, respectively, over the five days (n=60 injections Cyclosporin A
±20%
%RSD = 2.36
each) (Figure 4). Phospholipids showed no buildup over 0

the course of the study.

30
B
25

500,000 A Cyclosporin D in Whole Blood 20

Calculated ng/mL
15
4.0% RSD Day 1 Day 2 Day 3 Day 4 Day 5
Peak Area

10
250,000
5 Everolimus ±20%
%RSD = 4.22
0

0
30
C

B Ascomycin Peak Area %CV = 8.29% 25

90,000
Calculated ng/mL

20
70,000
15
Peak Area

50,000
Day 1 Day 2 Day 3 Day 4 Day 5
Day 1 Day 2 Day 3 Day 4 Day 5 10
30,000
5 Sirolimus
±20%
%RSD = 4.08
10,000 Ascomycin Peak Area ± 20%
0
1 145 290 435 580 725 870 1015 1160 1305 1452
Injection #
Figure 3. Raw peak area reproducibility across 5 days at 20 D
approximately 300 injections per day from different batches 18
of whole blood for (A) cyclosporin D internal standard and 16
(B) ascomycin internal standard. 14
Calculated ng/mL

12
10
8 Day 1 Day 2 Day 3 Day 4 Day 5
6
4
Tacrolimus ±20%
2 %RSD = 3.57
0
14 200 400 600 800 1000 1200 1464
Injection # (12/day)
Figure 4. Quality control precision for (A) cyclosporin A,
(B) everolimus, (C) sirolimus, and (D) tacrolimus, during 5-day
robustness testing. Twelve QCs per day were injected throughout each
day’s run of approximately 300 samples.

4
Conclusion
• The TSQ Quantis triple quadrupole MS demonstrated
robust, reliable, and consistent performance for more
than 1500 samples over five days with no need for
maintenance.

• Calibrators and controls maintain precision and

accuracy demonstrating reliability.

• The quantitative workflow exhibited reproducible results

on a sensitive and robust platform suitable for analysis
of immunosuppressant drugs in blood for clinical
research.

For Research Use Only. Not for use in diagnostic procedures.

Find out more at thermofisher.com/Altis-Quantis

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TN64969-EN 0517S