VISUALIZATION AND QUANTIFICATION OF DRUG/METABOLITES BY SENSITIVE AND FAST TARGETED DESI IMAGING ON TQ SYSTEM

Importance of the Topic

The spatial distribution and quantification of drugs and their metabolites within tissue are critical for understanding pharmacokinetics and pharmacodynamics. Traditional LC-MS methods require tissue homogenization, losing spatial context. Fast, sensitive desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on a triple quadrupole system overcomes these limitations, enabling targeted imaging and quantification of small molecules directly on tissue sections without radioactive labeling.

Objectives and Study Overview

This study aimed to evaluate a targeted DESI-MSI workflow on the Xevo TQ Absolute mass spectrometer for imaging the CNS drug pitolisant and its oxidized metabolite in mouse liver and brain. Key goals included determining limits of detection, optimizing acquisition speed, and demonstrating quantitative capabilities using an in-house MicroApp.

Methodology

1. Tissue and Standards Preparation

• Dilution series of pitolisant and metabolite (0.05–10 ng) spotted on control porcine liver and mouse brain sections.
• Mouse dosing at 5 mg/kg (therapeutic) and 75 mg/kg (NOAEL), organs harvested 2 h post-dose, frozen and cryosectioned (18 µm).

2. DESI-MSI Acquisition

• Instrument: Xevo TQ Absolute with DESI-XS source and High-Performance Sprayer.
• Spray: 2 µl/min, 95:5 MeOH:H₂O, 10 psi N₂, capillary voltage 0.55–0.7 kV (positive mode).
• Pixel sizes: 15 µm and 50 µm.
• MRM transitions: pitolisant (296.2 > 98.1, 296.2 > 126.15), metabolite (312.1 > 98.05), plus seven lipid markers.
• Acquisition speeds: 10 Hz for ten transitions, 50 Hz for two transitions (minimum dwell time 6 ms).

Used Instrumentation

• Xevo TQ Absolute mass spectrometer
• DESI-XS source with High-Performance Sprayer
• MassLynx v4.2 and High Definition Imaging (HDI) v1.8 software
• In-house MicroApp MSI Quantify for calibration and quantification

Results and Discussion

• Limits of detection reached 0.05 ng for both pitolisant and its oxidized metabolite on tissue.
• Fast imaging (average 43 min per large liver section) at 50 µm resolution detected drug and metabolite with signal-to-noise > 10 at both dose levels, including brain.
• Ten-transition imaging at 10 Hz provided simultaneous maps of drug, metabolite, and lipid biomarkers, revealing similar distributions at high dose and distinct intratissue localization at therapeutic dose (drug enriched near lobule, metabolite more diffuse).
• Quantitative MSI using calibration curves from the dilution series enabled estimation of drug concentration in defined ROIs on dosed tissue sections.

Benefits and Practical Applications

• Combines high sensitivity, specificity, and spatial resolution in a single workflow.
• Avoids homogenization and radioactive tracers, preserving tissue context.
• Rapid acquisition supports throughput for DMPK, QA/QC, and biomarker studies.
• Flexible MRM panel allows co-mapping of drugs, metabolites, and endogenous markers.

Future Trends and Opportunities

• Extension to other therapeutic classes and metabolite panels.
• Integration with high-resolution mass analyzers and ion mobility separations.
• Automated ROI detection and real-time quantitative reporting.
• Multimodal imaging combining MSI with histological or fluorescence data.
• Clinical tissue biopsy applications for personalized pharmacokinetics.

Conclusion

This work demonstrates that targeted DESI-MSI on a triple quadrupole platform provides sub-nanogram detection and fast imaging of drug and metabolite distributions in tissue. The quantitative workflow, supported by MicroApp MSI Quantify, offers a robust tool for spatial pharmacokinetics in research and industry.

Reference

1. Ligneau X., Landais L., Perrin D., et al. Biochemical Pharmacology. 2007;73(8):1234–1242.