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Quantitation of Immunosuppressant
Drugs in Whole Blood Using the Prelude-
SPLC System and TSQ Endura Mass
Spectrometer for Research
Bill Yu, Joe DiBussolo, Kristine Van Natta, Marta Kozak
Thermo Fisher Scientific, San Jose, CA
Application Note
604
Key Words
Immunosuppressant drugs, Prelude SPLC, TSQ Endura
Goal
To develop a rapid, sensitive, selective, and robust LC-MS/MS method to
determine the concentrations of cyclosporine A, tacrolimus, sirolimus, and
everolimus in whole blood.
Introduction
LC-MS/MS-based methods have advantages due to their
selectivity and low cost compared to traditional
immunoassay-based methods. In addition, unlike
immunoassays, LC-MS/MS-based methods are ideally
suited to the analysis of multiple compounds in a single
analytical run. In this report, a single analytical run was
used to precisely and accurately measure the levels of four
immunosuppressant drugs in blood for research. This was
accomplished by using a sample preparation and liquid
chromatography (SPLC)-MS/MS system, which combines
online sample extraction powered by Thermo Scientific™
TurboFlow ™ technology with chromatographic separation.
The system features
two independent channels of sample preparation and
liquid chromatography. Thus, the chromatographic
methods on the Prelude SPLC system can be executed in
parallel, either with a different method on each channel or
the same method on both channels. Two channel
multichannel operation on the Prelude SPLC system is
automatically optimized into one mass spectrometer for
serial detection, which improves mass spectrometer
utilization time, increases throughput, and reduces
analysis cost. The Prelude SPLC syringe pumps and high
pressure, low-volume gradient mixing provide enhanced
HPLC performance with improved peak shape and
resolution as well as stable retention times, compared
to the dual piston reciprocating pumps.
Methods
Sample Preparation
A 200 µL aliquot of whole blood sample was mixed with
300 µL of zinc sulfate solution (0.1 M) in a 1.5 mL
centrifuge tube and vortexed for 30 seconds. The mixture
was further processed by adding 500 µL of
containing internal standards
(40 ng/mL D12-cyclosporine A and 4 ng/mL 13CD2tacrolimus).
The sample was immediately vortexed for another
30 seconds. The entire mixture was centrifuged at
4000 RCF for 10 minutes. A 40 µL sample was analyzed.
Liquid Chromatography
SPLC-MS/MS analysis was conducted using a Prelude
SPLC powered by TurboFlow technology coupled to a
riple quadrupole mass
spectrometer. The processed sample was directly injected
onto a Thermo Scientific™ Cyclone-P™ column (0.5 x 50 mm,
Part Number: CH-953289) for online sample cleanup. This
step was followed by chromatographic separation on a
3 x 30 mm,
2.6 µm particle size, Part Number: 17226-033030). The
Cyclone-P TurboFlow column was maintained at room
temperature while the Accucore C8 column was
maintained at 70 °C. The total run time was 5 minutes
and the total solvent consumption was 8.1 mL per
sample, including online sample extraction and
chromatographic separation. Figure 1 shows the SPLC
method profile.