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Introduction Melia toosendan Sieb. et Zucc. has been used as a Chinese folk medicine for roundworm treatment since ancient times. Many diverse limonoids have been isolated from Meliaceae plants, but it remains difficult to isolate and identify other limonoids because of their small natural concentrations. Objective This study was performed to overcome the difficulties associated with fast and accurate identification of limonoids and establish a reliable and sensitive method for the analysis of minor limonoids in M. toosendan fruits. Methods An efficient strategy for enrichment, detection, and identification of minor limonoids from M. toosendan fruits using solid‐phase extraction with high‐performance liquid chromatography–quadrupole time‐of‐flight tandem mass spectrometry (SPE‐HPLC‐Q‐TOF‐MS/MS) was developed herein. Results Characteristic fragmentations and fragmentation ions containing trichilin‐, nimbin‐, and vilasinin‐class limonoid skeletons were initially studied, and characteristic diagnostic ions involved retro Diels‐Alder (RDA) reactions or homolytic cleavages, which were used to identify minor limonoids. In total, 13 limonoids, including four new ones, were identified. Conclusion This is the first report on the analysis of M. toosendan fruits to identify limonoids. This novel analysis method may stimulate further research regarding the identification of limonoids in other plant species.

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