Abstract
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.
Highlights
D etermining physiological distributions of drugs in tissues plays an important role in pharmaceutical research, toxicology, and medicine but is often difficult
We report a comprehensive validation of a quantitative protocol for MALDI-MSI analysis of distributions of a drug in the brain using instruments with either time-of-flight (TOF) or Fourier transform ion cyclotron resonance (FTICR) mass analyzers
Citalopram, a lipophilic selective serotonin reuptake inhibitor that readily passes through the blood−brain barrier and widely distributes in the brain,[14] was administered in vivo as a model substance to mice, and brain tissue was collected for analysis
Summary
D etermining physiological distributions of drugs in tissues plays an important role in pharmaceutical research, toxicology, and medicine but is often difficult. Ligand binding assays are widely applied for quantitative measurements of analytes in biological samples.[1] during the sample preparation required for LC−MS/MS analyses, information about biomolecules and drug spatial distributions in tissues is inevitably lost. Quantitative whole-body autoradiography (QWBA) ligand-binding assays provide spatial information but have less molecular specificity than MS because both analytes and metabolites may contain the radioactive label. Different MSI strategies have been studied for the quantitative imaging of drugs directly in tissue sections.[3] Three different methods have been exploited for applying calibration standards and internal standards: (i) the dilution series model,[4] (ii) tissue extinction coefficient model,[5] and (iii) mimetic tissue model.[6] The dilution series model has been most commonly used because it is relatively straightforward and takes less time to prepare.[3]
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