MULTI-STEP WORKFLOW FOR VISUALIZATION OF DRUG/METABOLITES AND METABOLISM WITH DESI TQ AND DESI Q-TOF MASS SPECTROMETERS

Importance of the Topic

Desorption electrospray ionization mass spectrometry imaging (DESI MSI) provides direct spatial mapping of pharmaceutical compounds, their metabolites and endogenous molecules in tissue sections without prior homogenization.

Objectives and Study Overview

This study implements a multi-step DESI MSI workflow to visualize the distribution and temporal metabolism of the tyrosine kinase inhibitor Gefitinib and 16 of its known metabolites in mouse liver sections collected at 0.5, 1, 3, 8 and 24 hours post dose.

Methodology and Instrumentation

Tissue Preparation and Dosing

• Male C57BL/6 mice received an IV tail‐vein dose of Gefitinib (10 mg/kg).
• Livers were harvested at specified time points, flash-frozen and cryo-sectioned at 18 µm.

DESI MSI Workflow

• Full scan imaging on a SELECT SERIES™ MRT (Q-ToF) with mass resolution > 200 000 FWHM and < 500 ppb accuracy for untargeted discovery.
• Targeted MRM imaging on a tandem quadrupole (TQ) MS for enhanced sensitivity and specificity using previously optimized MRM transitions for Gefitinib, 16 metabolites and potassiated PC(34:1).
• High-Performance Sprayer (HPS) on DESI XS source to improve spray focus and signal intensity.

Data Processing

MassLynx™ and HDI™ software were used to define regions of interest (ROIs) and extract normalized signal intensities, which were then analyzed by MetaboAnalyst for multivariate statistics and temporal profiling.

Key Results and Discussion

Untargeted Discovery

• Gefitinib was localized in liver tissue up to 8 hours, with signal declining thereafter; no metabolites were detected in full-scan mode.
• PCA of endogenous and drug-derived signals clearly clustered samples by time post dose, indicating metabolic phenotype changes.

Targeted MRM Imaging

• Ten of the 16 known Gefitinib metabolites were detected in dosed livers but absent in controls.
• Metabolites M7, M9, M10, M11, M12 and M14 peaked at 0.5 h post dose; M1, M2, M6 and M13 maximized at 3 h.
• Potassiated PC(34:1) lipid signal was highest at 0.5 h, decreased at 1 h, then partially recovered over time.

Benefits and Practical Applications of the Method

• Combining high-resolution full scan and targeted MRM enables both discovery and sensitive quantitation in a single workflow.
• Direct tissue imaging preserves spatial information critical for understanding drug distribution and localized metabolism.
• Applicable to DMPK studies, toxicology, QA/QC and pharmaceutical research to accelerate in situ chemical phenotyping.

Future Trends and Opportunities

• Integration of quantitative MSI approaches to provide absolute concentration maps.
• Expansion to 3D imaging and higher spatial resolution for subcellular localization.
• Incorporation of artificial intelligence for automated feature extraction and classification.
• Multimodal imaging combining DESI with complementary techniques (e.g., MALDI, optical microscopy).

Conclusion

The presented multi-step DESI MSI workflow successfully visualizes Gefitinib distribution, resolves sixteen metabolites with high specificity, and reveals time-dependent metabolic alterations directly in liver tissue, demonstrating its value for advanced DMPK and metabolomic studies.

Reference

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