Page 127 - CRFTApplication Compendium_090613

Quantitation of Immunosuppressant Drugs
in Blood Using a Second-Generation
High-Resolution, Accurate-Mass Mass
Spectrometer
Kristine Van Natta and Marta Kozak
Thermo Fisher Scientific, San Jose, CA
Application Note
577
Key Words
Immunosuppressant, tacrolimus, sirolimus, everolimus, cyclosporine A,
Exactive Plus, TraceFinder, ClinSpec, clinical research, FK-506
Goal
Evaluate the combined use of a second-generation Thermo Scientific™
Orbitrap™-based high-resolution mass spectrometer and an
immunosuppressants test kit for clinical research analysis of
immunosuppressant drugs in whole blood. Apply the method to analysis
of samples previously analyzed using a validated method on a triple-stage
quadrupole mass spectrometer and compare the results.
Introduction
The Thermo Scientific™ ClinSpec™ Immunosuppressant
Test kit was developed for clinical research liquid
chromatography/tandem mass spectrometry (LC-MS/MS)
analysis of tacrolimus, sirolimus, everolimus, and
cyclosporine A in whole blood specimens. The kit consists
of six different calibrator levels, up to five quality control
levels, internal standard and extraction reagent. Here, the
kit is used with a second-generation high-resolution,
accurate-mass (HR/AM) mass spectrometer to analyze for
these compounds. The results are compared to results
previously obtained using a validated method and a
triple-stage quadrupole mass spectrometer.
Experimental
Sample Preparation
Samples were prepared per the package insert in the
ClinSpec kit.1 Briefly, whole blood samples were processed
by precipitation with ZnSO4/methanol solution containing
internal standards ascomycin and cyclosporine D. The
samples were shaken for 30 minutes at room temperature
before being centrifuged at 13,000 rpm for 10 minutes.
The supernatant was transferred to an autosampler vial,
capped, and 50 µL was injected onto the HPLC system.
Liquid Chromatography
Chromatographic separation was performed using a
Thermo Scientific ™ Accela ™ 600 HPLC pump and Thermo
Scientific ™ Hypersil GOLD™ Javelin™ guard column, (10 x
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 water and methanol,
respectively. Mobile phase C was acetonitrile/1-propanol/
acetone (45:45:10). The total run time was 2 minutes.
Mass Spectrometry
Samples were analyzed with a Thermo Scientific™ Exactive™
Plus high-performance benchtop mass spectrometer equipped
with an Orbitrap mass analyzer. An atmospheric pressure
chemical ionization (APCI) probe was used as an ion
source. The instrument was operating in positive full-scan
mode at a resolution of 70,000 (FWHM) at m/z 200.
Relevant scan and source parameters are shown in
Tables 1 and 2.
Table 1. Scan parameters for Exactive Plus mass spectrometer
Scan Parameter Value
Mass range: m/z 800–4000
Resolution: 70,000
Polarity: Positive
Microscans: 1
Lock mass: Off
AGC target: 1 x 106
Max inject time: 200 msec