Tandem Quadrupole DESI Imaging To Visualize Spatial Distribution of Gefitinib and Related Metabolites in Rat Liver

Significance of the topic

The study demonstrates application of targeted desorption electrospray ionization tandem mass spectrometry (DESI-MS/MS) performed in multiple-reaction monitoring (MRM) mode on a tandem quadrupole instrument to map the spatial distribution of the tyrosine kinase inhibitor gefitinib and its metabolites in fresh-frozen rat liver. Mapping drug and metabolite localization without radiolabels or antibody tagging enables direct spatial pharmacokinetic and disposition information during discovery and early development, which is critical for understanding target engagement, local toxicity, and tissue-specific metabolism.

Objectives and study overview

• Evaluate targeted DESI MRM imaging on a Waters Xevo TQ-Absolute XR tandem quadrupole to detect and spatially map gefitinib and related metabolites in rat liver following a single therapeutic subcutaneous (SC) dose (10 mg/kg).
• Compare DESI imaging signal intensity and temporal distribution with orthogonal LC-MS/MS pharmacokinetics (plasma) and urine elimination data.
• Demonstrate feasibility of label-free spatial metabolomics at non-lethal dosing levels relevant to typical pharmacology studies.

Methodology

• Animal dosing and sampling: Male Wistar rats received a single 10 mg/kg SC dose of gefitinib. Plasma and urine were collected periodically up to 24 h for PK and metabolite identification. Fresh-frozen liver samples were collected at multiple post-dose time points (including 0.5, 1, 3, 6, 8 and 24 h) from four animals for DESI imaging.
• Tissue preparation: Livers were cryosectioned at 10 µm thickness and thaw-mounted onto standard glass slides; samples were stored at −80 °C and thawed briefly in a vacuum desiccator before analysis.
• DESI imaging: Targeted MRM imaging used a DESI source with 98:2 methanol:water containing 0.01% formic acid as spray solvent, flow rate 2 µL/min, emitter voltage +0.65 kV, sprayer pressure 0.9 bar, source temperature 120 °C, cone voltage 13–22 V. MS1/MS2 resolution ~0.75 Da FWHM. An internal standard (gefitinib-d6) was applied for signal normalization/quality control.
• Complementary LC-MS/MS: Plasma and urine analyses used ACQUITY Premier UPLC with an ACQUITY Premier BEH C18 column (2.1×100 mm, 1.7 µm). Sample prep: plasma and urine protein precipitation (5 µL, 4:1 acetonitrile:methanol for plasma), and urine solid-phase extraction using Oasis HLB for metabolite identification and quantitation. Targeted LC-MRM data were processed with TargetLynx XS; Skyline and MetaboAnalyst 6.0 were used under open licenses for data handling/visualization.
• Target selection: MRM transitions were designed to monitor precursor > diagnostic fragment (“fingerprint fragment”) pairs for gefitinib and six metabolites (denoted M2, M5, M6, M7/M9, M11, M12/M13 etc.), enabling selective MS/MS identification during imaging.

Used instrumentation

• Mass spectrometer: Waters Xevo TQ-Absolute XR tandem quadrupole MS configured for MRM imaging (DESI XS interface).
• Chromatography: ACQUITY Premier UPLC system with BEH C18 column (2.1×100 mm, 1.7 µm).
• Sample preparation: Oasis HLB SPE plates/cartridges and standard protein precipitation reagents (acetonitrile:methanol).
• Software: TargetLynx XS for LC-MRM processing; HDI (for imaging workflows) and open-license tools Skyline and MetaboAnalyst 6.0 for downstream analysis.

Main results and discussion

• Detection and spatial mapping: DESI MRM imaging successfully detected gefitinib and six metabolites in fresh-frozen liver sections at a therapeutic 10 mg/kg dose. The method delivered sufficient sensitivity to observe metabolites without administering radiolabeled compound or using supratherapeutic doses.
• Temporal patterns: Most analytes (drug and metabolites) produced the highest liver signal intensity around 1 h post-dose and remained detectable at 24 h. The hepatic imaging intensities for gefitinib and selected metabolites (notably M11) correlated qualitatively with plasma pharmacokinetic profiles derived from LC-MS/MS.
• Correlation with LC-MS/MS: Quantitative/temporal trends from LC-MRM plasma concentrations and urine elimination profiles matched the spatial intensity patterns obtained by DESI imaging, supporting the biological relevance of the imaging readouts.
• Analytical selectivity: Using MRM transitions (precursor → fingerprint fragment) on a tandem quadrupole provided targeted MS/MS confirmation during imaging, increasing selectivity compared to simple precursor-only imaging and reducing false positives from isobaric interferences.
• Operational considerations: The ambient nature of DESI allowed minimal sample prep and rapid analysis of tissue sections. Method parameters (spray composition, flow, voltage, cone settings) were optimized to balance ionization efficiency and spatial fidelity.

Benefits and practical applications

• Label-free spatial pharmacokinetics: Enables mapping of parent drug and metabolites in tissue microenvironments without radiolabels, facilitating early DMPK and toxicology studies when radiolabeled material is unavailable.
• Reduced sample preparation and throughput: DESI imaging is ambient and fast compared with autoradiography or extensive tissue extraction workflows, allowing efficient screening of multiple time points or treatment groups.
• Integration with LC-MS/MS: Combining imaging on the same tandem-quadrupole platform with orthogonal LC-MRM workflows provides cross-validated spatial and quantitative pharmacokinetic information, strengthening interpretation for lead optimization and safety decisions.
• Regulatory and discovery utility: Spatial metabolomics data can inform on-site-specific metabolism, potential on-target/off-target exposure, and help prioritize follow-up studies (e.g., histopathology co-registration).

Future trends and applications

• Quantitative imaging: Development of robust tissue calibration strategies and isotopically labeled imaging standards to transition DESI-MRM from qualitative/semiquantitative maps to fully quantitative tissue concentration maps.
• Higher spatial resolution: Advances in DESI sprayer design and stage control to approach cellular or subcellular resolution, enabling finer-scale localization of metabolites within tissue microarchitecture.
• Multimodal integration: Co-registration of DESI images with histology, MALDI imaging, and other modalities (e.g., IMS, autoradiography) to combine molecular, morphological and functional information.
• Instrument coupling: Hybrid workflows combining tandem quadrupole MRM sensitivity with ion mobility or high-resolution MS for improved isomer separation and confident metabolite identification.
• Data analytics: Application of machine learning and advanced statistical frameworks to extract spatial patterns, relate them to PK/PD endpoints, and predict sites of metabolism or toxicity.
• Translation to clinical and regulatory settings: Methods to analyze human biopsy or surgical tissue for drug distribution and local metabolism, aiding translational pharmacology and personalized medicine.

Conclusion

Targeted DESI MRM imaging on a tandem quadrupole instrument provided sensitive, label-free spatial mapping of gefitinib and multiple metabolites in rat liver at therapeutically relevant dosing. The imaging results correlated with LC-MS/MS plasma and urine pharmacokinetics, supporting the method as a practical tool for early-stage spatial DMPK assessment. Continued improvements in quantitation, spatial resolution, multimodal integration and data analytics will expand the utility of DESI MRM imaging in drug discovery and translational studies.

References

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