Integrated phytohormone quantification and metabolomics analysis enables new insights into the soil phytohormonome

The contribution of soil chemistry to plant growth and resilience, including presence of phytohormones, is increasingly recognized. However, comprehensive characterization of soil phytohormones remains limited by chemical complexity of soil matrices, diversity and low- abundance of metabolites. To enable further discoveries we developed and validated performance of a liquid chromatography-mass spectrometry method with solid phase extraction, integrating targeted and untargeted hormonomic approaches for comprehensive soil phytohormone profiling. Method performance was evaluated for sixteen plant growth-regulating compounds and precursors, including abscisic acid, auxins, cytokinins, gibberellic acid, jasmonic acid, salicylic acid, karrikins, melatonin, serotonin, and tryptophan. The method demonstrated strong linearity (R{superscript 2} = 0.989-0.999), high sensitivity (limits of detection and quantification 0.1-50.2 and 1.4-167.3 pg on-column, respectively), and acceptable precision (1.3-9.6% intraday; 3.4-34.8% interday). Soil composition had a significant effect on recovery, with recovery being poor in some soils such as clay-rich soils; however, recovery for most phytohormones were within 20% of the matrix- adjusted spiked value. Validation results confirm that the method is suitable for use and was then used to quantify analytes in representative soil types. Integration of untargeted analysis expanded coverage to 250 additional putative phytohormones and hormone-related metabolites, revealing chemical signatures potentially associated with plant community composition. The method is robust across these soils spanning sandy, peat-rich, and clay-rich textures. This approach provides a versatile framework for investigating belowground phytohormone dynamics and their roles in plant physiology, resilience, and soil-plant feedbacks.