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Application Note 618 An LC-MS/MS Research Method for the
Quantification of Mycophenolic Acid (MPA)
in Plasma
Pascal Guérard1, Maeva Wendremaire1, Bénédicte Duretz 2
1Pharmacology and Toxicology Laboratory, Dijon Hospital, Dijon, France
2Thermo Fisher Scientific, Les Ulis, France
Key Words
TSQ Quantum Ultra, mycophenolic acid, MPA, plasma, quantitation
Goal
The goal of this work was to use LC-MS/MS to validate the MassTox®
Mycophenolic Acid kit from ChromSystems® on th
for research purposes.
Introduction
This note describes a method developed to quantify
mycophenolic acid (MPA) by LC-MS/MS with the
ChromSystemsMassTox Mycophenolic Acid kit. The
method was analytically validated for research use using
the following parameters:
• Both intraday and interday accuracy and precision for
the quality controls
• Lower limit of quantitation (LLOQ) and upper limit of
quantitation (ULOQ)
• Carryover
Methods
The MassTox Mycophenolic Acid kit consists of:
• Four calibrators (1 blank and 3 calibrators)
• Two quality controls (Level I and Level II)
• Internal standard set consisting of an internal
standard mix and a reconstitution buffer
• Precipitation reagent solution
• Extraction buffer solution
• Mobiles phases A and B
Calibrator and Quality Control (QC) Preparation
Lyophilized calibrators and quality controls were
reconstituted with 1 mL of distilled water. They were
left at room temperature for 15 minutes and shaken
occasionally until the contents were homogeneous.
Aliquots of 50 µL were stored in 1.5 mL vials at -20 °C
for a maximum period of 3 months.
Internal Standard Preparation
Internal standards were reconstituted with 1 mL of
reconstitution buffer. The vial was left for 5 minutes at
room temperature. It was shaken periodically and gently
until the contents were homogeneous. Next, 800 µL of this
solution was added to 12 mL of precipitation reagent and
the mixture was stored in the dark at 4 °C for 28 days.
Sample Preparation
A 25 µL measure of extraction buffer was added to 50 µL
of each calibrator, control, and sample. The mixture was
vortexed for 10 seconds and incubated for 2 minutes at
room temperature. Then, 250 µL of reconstituted internal
standard mix was added to the vial and vortexed for
30 seconds. It was then centrifuged at 14,000 rpm for
5 minutes. Finally, 10 µL of the supernatant was diluted
20 times in a mixture containing methanol and water
(LC/MS grade) (50/50, v/v).
Calibration Curve
The concentrations of the calibrators were 0.97, 3.89,
and 9.46 mg/L.