Cross-validated Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Quantitation Protocol for a Pharmaceutical Drug and Its Drug-Target Effects in the Brain Using Time-of-Flight and Fourier Transform Ion Cyclotron Resonance Analyzers

Matrix-assisted laser desorption/ionization
mass spectrometry imaging
(MALDI-MSI) is an established tool in drug development, which enables
visualization of drugs and drug metabolites at spatial localizations
in tissue sections from different organs. However, robust and accurate
quantitation by MALDI-MSI still remains a challenge. We present a
quantitative MALDI-MSI method using two instruments with different
types of mass analyzers, i.e., time-of-flight (TOF) and Fourier transform
ion cyclotron resonance (FTICR) MS, for mapping levels of the in vivo-administered drug citalopram, a selective serotonin
reuptake inhibitor, in mouse brain tissue sections. Six different
methods for applying calibration standards and an internal standard
were evaluated. The optimized method was validated according to authorities’
guidelines and requirements, including selectivity, accuracy, precision,
recovery, calibration curve, sensitivity, reproducibility, and stability
parameters. We showed that applying a dilution series of calibration
standards followed by a homogeneously applied, stable, isotopically
labeled standard for normalization and a matrix on top of the tissue
section yielded similar results to those from the reference method
using liquid chromatography–tandem mass spectrometry (LC–MS/MS).
The validation results were within specified limits and the brain
concentrations for TOF MS (51.1 ± 4.4 pmol/mg) and FTICR MS (56.9
± 6.0 pmol/mg) did not significantly differ from those of the
cross-validated LC–MS/MS method (55.0 ± 4.9 pmol/mg).
The effect of in vivo citalopram administration on
the serotonin neurotransmitter system was studied in the hippocampus,
a brain region that is the principal target of the serotonergic afferents
along with the limbic system, and it was shown that serotonin was
significantly increased (2-fold), but its metabolite 5-hydroxyindoleacetic
acid was not. This study makes a substantial step toward establishing
MALDI-MSI as a fully quantitative validated method.