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Quantitation of Mycophenolic Acid
in Plasma for Research by TurboFlow
HPLC-MS/MS
Neil Leaver
1,2
, Sarah Robinson
3
1
Royal Brompton & Harefield NHS Foundation Hospital, Harefield, UK
2
Imperial College, London, UK
3
Thermo Fisher Scientific, Hemel Hempstead, UK
Application Note 612
Key Words
Mycophenolic acid, TurboFlow online extraction method,
mass spectrometry, APCI, therapeutic drug monitoring in research,
MPA, immunosuppressants, organ transplantation
Goal
To present a precise and accurate research method for the
quantitation of total mycophenolic acid (MPA) in plasma using
Thermo Scientific
™
TurboFlow
™
technology.
Introduction
Mycophenolic acid (MPA) is an immunosuppressive drug
commonly used in solid organ transplantation. MPA
has been available since the early 1990s, but the merits
of monitoring MPA levels in blood plasma have still not
been finalized.
1
Many research laboratories utilize plasma
concentrations from commercial assays instead of
generating their own analytical methods. Some published
methods show MPA analyzed along with cyclosporine,
tacrolimus, sirolimus, and everolimus. However,
combining the analytes into one method is not advisable
because MPA is measured in plasma and the others are
measured in whole blood, which would require separate,
matrix-matched calibrations. It is therefore better to have
a simple and fast method for MPA analysis.
To accurately measure MPA with high throughput and
minimal sample preparation, a method using
LC-MS/MS and TurboFlow technology has been
developed. All validation was performed in compliance
with current guidelines.
2,3
Experimental
Reagents
Three mobile phases were required for the
TurboFlow method (all Fisher Chemical brand reagents):
Mobile phase A:
100% ultrapure water +
10 mM ammonium acetate
(Must be made fresh daily, unless an
antimicrobial reagent such as 2% acetonitrile or
5% methanol is added.)
Mobile phase B:
100% methanol + 10 mM ammonium acetate
Mobile phase C:
Acetonitrile/2-propanol /acetone
(40:40:20 v/v/v) (used for cleaning the
TurboFlow column and a wash solution for
the autosampler)
Autosampler wash 1: 100% water (no modifier)
Autosampler wash 2: Mobile phase C
Extracting reagent:
Zinc sulphate in methanol
Sample Preparation
Plasma samples were separated from EDTA-
anticoagulated whole blood within 24 hours of
venipuncture to prevent potential degradation of
mycophenolic acid glucuronide (MPAG) to MPA. Plasma
samples were stored at 4–8 °C while awaiting analysis.
Plasma samples, calibrators (Chromsystems Instruments
& Chemicals GmbH, Germany), and quality controls
(More Diagnostics, Inc, CA, USA) were extracted by
adding 100 µL sample, 100 µL internal standard solution
(indomethacin), and 800 µL extracting solution. This was
vortex-mixed and centrifuged for 5 minutes at >10,000
g
.
Supernatants were transferred to a microtitre plate and
covered with a silicon sealing mat before being loaded
into the autosampler thermostatted at 10 °Cwhile
awaiting analysis.