Development and Validation of a Targeted LC-MS/MS Method for Simultaneous Quantification of Allelochemicals in Root Exudates

Allelopathy mediates below-ground competitive interactions between rice and weeds in paddy ecosystems. Diverse phytotoxic allelochemicals complicate their simultaneous quantification in root exudates. This study aimed to develop a targeted LC-MS/MS method for profiling fourteen allelochemicals spanning five classes. Multiple reaction monitoring (MRM) transitions were optimized for reference standards, and method performance was validated for linearity, sensitivity, precision, recovery, and matrix effects. All analytes exhibited good linearity (R2 > 0.99), with limits of detection (LODs) of 0.005–0.641 ng/mL and limits of quantification (LOQs) of 0.017–2.136 ng/mL. Intra- and inter-day precision ranged from 0.27–4.90% and 0.45–4.51%, respectively, while matrix effects were 86.21–113.99%. In the root exudates of rice, Echinochloa crus-galli, Leptochloa chinensis, and sorghum, caffeic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid, loliolide, and DMBQ were detected in all species, whereas the other analytes exhibited species-specific distribution patterns. Notably, momilactone A was only detected in Nipponbare (NIP) rice and E. crus-galli root exudates. Principal component analysis (PCA) based on 11 detectable allelochemicals revealed distinct profiles among the four hydroponically cultured species. This validated LC-MS/MS platform enables characterization of allelochemical compositions in paddy root exudates.