Structural and quantitative analysis of plant hormone and its metabolites with LC-MS/MS

Significance of the topic

The plant hormone indole-3-acetic acid (IAA) orchestrates key processes in growth, development and stress responses. In living tissues, IAA exists largely as conjugates or oxidized forms, which serve not only as intermediates in metabolic turnover but also as reversible storage or transport pools. Sensitive and selective profiling of free and bound IAA species is critical for understanding hormone dynamics, pathway regulation and agronomic traits such as root architecture or stress tolerance.

Objectives and study overview

This study aimed to establish a robust liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) workflow combining quantitative multiple reaction monitoring (MRM) with precursor-ion and neutral-loss scanning. The goals were to quantify a broad panel of IAA metabolites in rice seedlings and to screen for novel or low-abundance conjugates in a single analytical run.

Methodology and instrumentation

• Sample preparation: Two-week-old Oryza sativa cv. Nipponbare seedlings were grown hydroponically in water or 10 µM IAA. Aerial and root tissues were flash-frozen, ground under liquid nitrogen, and extracted with acetone–water (4:1) containing 2.5 mM diethyldithiocarbamic acid. The combined extracts were concentrated, partially purified on C18 cartridges and reconstituted for analysis.
• Chromatography: Shimadzu Nexera UHPLC with Cadenza CD-C18 column (75 mm × 2 mm, 3 µm) at 30 °C. Mobile phases were 0.05% acetic acid in water (A) and methanol (B), with a linear gradient from 10% to 90% B over 15 min at 0.2 mL/min. Injection volume was 3 µL.
• Mass spectrometry: Shimadzu LCMS-8030 triple quadrupole with rapid polarity switching (15 ms), dwell and pause times of 1 ms, and scan speed up to 15,000 u/sec. MRM transitions were optimized for IAA, IAA-Asp, oxidized IAA (OxIAA), dihydro-OxIAA (DiOxIAA) and their Asp/Glu and glucose conjugates. Precursor-ion scans at m/z 146(+) and 130(+), and neutral-loss scans of 161(+) and 162(−) were employed for untargeted metabolite screening.

Main results and discussion

• Calibration linearity: All targeted metabolites showed linear responses from 1 to 1,000 nM (r² > 0.999).
• Quantitative profiling: In IAA-treated seedlings, concentrations of IAA-Asp, OxIAA, DiOxIAA and related conjugates increased substantially compared to controls, with root levels consistently higher than in aerial tissues. For example, IAA-Asp reached ~780 nM in roots versus <1 nM in controls.
• Peak quality: High-speed data acquisition afforded >10 points per peak even with simultaneous MRM and screening scans, yielding well-shaped chromatographic profiles.
• Metabolite discovery: Precursor-ion and neutral-loss scanning in root extracts confirmed six known conjugates and indicated additional candidate ions for further structural characterization.

Benefits and practical applications

The single-run method streamlines quantitative and qualitative analysis of hormone metabolites, reducing sample consumption and instrument time. It offers high sensitivity, broad dynamic range and rapid polarity switching, ideal for plant physiology studies, breeding screens, stress response profiling and quality control of botanical products.

Future trends and applications

Advances may include high-resolution MS for exact mass confirmation, integration with imaging-MS for spatial mapping, automated data processing for large-scale phenotyping, and extension to other phytohormone classes. Coupling metabolomics with transcriptomics will deepen insights into regulatory networks.

Conclusion

This work demonstrates a powerful LC-ESI-MS/MS platform capable of precise quantitation and comprehensive screening of IAA metabolites in plants. The combined MRM and scanning approach enhances throughput and discovery potential in hormone research.

References

• K. Kai et al., Phytochemistry 68 (2007) 2512–2522
• K. Kai et al., Phytochemistry 68 (2007) 1651–1663