Multidirectional Analysis of Plant Alkaloids Using MS Imaging and OAD-TOF System

Significance of the topic

Mass spectrometry imaging (MSI) combined with advanced MS/MS fragmentation strategies enables spatially resolved chemical characterization in biological tissues. For plant alkaloids—bioactive, often toxic nitrogen-containing secondary metabolites—knowing both tissue distribution and molecular structure is essential for pharmacology, food safety, plant biology, and natural product discovery. Integrating MSI with novel radical-driven fragmentation (Oxygen Attachment Dissociation, OAD) alongside conventional Collision-Induced Dissociation (CID) improves structural assignment confidence and facilitates identification of known and unknown alkaloids.

Objectives and study overview

The study aimed to demonstrate a multidirectional workflow that couples imaging mass spectrometry (iMScope QT) with an OAD-enabled TOF MS/MS system to (1) map the spatial distribution of potato glycoalkaloids in sprouted tuber tissue and (2) improve structural elucidation by combining OAD-MS/MS with CID-MS/MS and in silico candidate screening.

Methodology

Plant material: Commercial Solanum tuberosum tubers were sprouted under sunlight; sprouts were frozen and cryosectioned at 10 μm thickness and mounted on ITO-coated glass slides.

Matrix deposition and MSI: α-Cyano-4-hydroxycinnamic acid (CHCA) was deposited using the iMLayer system to a nominal thickness (~0.7 μm). MSI acquisitions used the iMScope QT with spatial resolutions of 25 μm (whole section) and 10 μm (optical microscope region with 5× objective). Laser parameters were varied (diameter settings 1/2/4, repetition frequency up to 100 Hz) and typical laser intensities were applied to optimize signal.

MS/MS structural analysis: An OAD-TOF system was used to generate O/OH• radicals from a water vapor–hydrogen gas mixture in a microwave-driven radical source, introducing neutral radicals into the Q2/OAD collision cell. OAD-MS/MS spectra were acquired alongside CID-MS/MS spectra (low and high collision energies) to capture complementary fragmentation routes. Data analysis combined IMAGEREVEAL MS for imaging and LabSolutions Insight Explore for MS/MS interpretation and in silico fragment prediction; candidate structures were cross-checked against ChemSpider.

Instrumentation used

• Imaging mass microscope: iMScope QT (Shimadzu)
• OAD-enabled time-of-flight system: OAD-TOF (radical source providing O/OH•)
• Quadrupole TOF LCMS-9050 platform (OAD radical source illustrated on LCMS-9050)
• Matrix deposition: iMLayer
• Software: IMAGEREVEAL MS for MSI visualization; LabSolutions Insight Explore for MS/MS interpretation and in silico fragment generation

Main results and discussion

Distribution imaging: MSI of potato sections revealed strong localization of glycoalkaloids—solanine (m/z 868.508 [M+H]+) and chaconine (m/z 852.513 [M+H]+)—concentrated in sprouts and adjacent skin tissue, consistent with their defensive role in plants.

Structural elucidation: Treating solanine as an unknown, CID-MS/MS spectra were compared to in silico predicted fragments to produce structural candidates. Several isomeric or related structures matched formula-based constraints; however, CID alone could not uniquely assign the structure. OAD-MS/MS provided a diagnostic fragment at m/z 150.128 detectable at low collision energy that was absent in CID spectra. Accurate mass and elemental composition of this OAD-specific fragment (interpreted as C10H16N) and its formation via preferential cleavage near heteroatoms allowed exclusion of alternative candidates and supported assignment to solanine. Thus, OAD supplied orthogonal, diagnostic fragments—particularly useful for locating heteroatom-adjacent cleavages and C=C positional information—complementing CID fragmentation patterns.

Benefits and practical applications

• Improved identification confidence: Combining radical-driven OAD with CID gives complementary fragmentation fingerprints that narrow candidate structures, important when standards are unavailable.
• Spatially resolved chemistry: MSI maps distribution of bioactive alkaloids within plant tissues, informing studies of plant defense, toxicity hotspots (food safety), and metabolic compartmentalization.
• Discovery potential: The workflow supports discovery of novel alkaloid analogs by linking localization patterns with structural clues from OAD diagnostics.
• Applicability: Relevant for metabolomics, natural product research, pharmaceutical lead discovery, agricultural chemistry, and quality control in food sciences.

Future trends and potential applications

Expected developments and opportunities include:

• Higher spatial and mass resolution MSI combined with OAD to resolve subcellular localization and isomeric compounds.
• Broader adoption of radical-driven dissociation chemistries (OAD and related approaches) integrated into routine structural workflows and spectral libraries for automated annotation.
• Integration with advanced in silico tools, machine learning, and expanded databases (including OAD-specific fragment libraries) to accelerate unbiased natural product identification.
• Quantitative MSI improvements and multimodal imaging (e.g., combining MSI with microscopy or MALDI-IMS correlates) to link chemistry to histology or physiology.
• Application to diverse biological matrices beyond plant tissue, including microbial, environmental, and clinical specimens where radical fragmentation can reveal otherwise ambiguous structural features.

Conclusion

The combined use of iMScope QT imaging and an OAD-capable TOF MS/MS system provides a robust multidirectional approach for mapping and structurally characterizing plant alkaloids. OAD supplies diagnostic radical-based fragments that complement CID spectra, enabling more confident structural assignments—demonstrated here for solanine in potato sprouts. This integrated workflow enhances discovery and identification of biologically relevant alkaloids and can be extended to broader metabolomic and natural-product investigations.

References

1. Takahashi, H.; et al. Analytical Chemistry 2018, 90 (12), 7230–7238.
2. Takahashi, H.; et al. Mass Spectrometry. 2019, S0080.
3. Uchino, H.; et al. Communications Chemistry 2022, 5, 162.