An Integrated Approach for the Comprehensive Characterization of Metabolites in Broccoli (Brassica oleracea var. Italica) by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

BACKGROUND

Broccoli contains diverse phytochemicals, including glucosinolates and their hydrolysis products, with potential nutritional and bioactive properties. Accurate metabolite profiling requires optimized sample preparation and comprehensive databases.

METHODS

A rapid enzymatic deactivation method with 70% methanol, implemented prior to cryogrinding, was evaluated for processing freeze-dried and fresh broccoli florets, which were compared as plant materials. A widely targeted, organ-resolved metabolite database was constructed by integrating over 612 reported phytochemicals with glucosinolate degradation products. LC-HRMS combined with MS-DIAL and GNPS was employed for metabolite detection and annotation.

RESULTS

Freeze-dried samples yielded nearly twice the number of glucosinolates, isothiocyanates, and nitriles compared with standard-processed fresh tissue. Methanol pre-treatment preserved metabolite integrity in fresh samples, achieving comparable sensitivity to freeze-dried material. Using the integrated database, 998 metabolites were identified or tentatively characterized, including amino acids, carboxylic acids, phenolics, alkaloids, terpenoids, and glucosinolate derivatives. Cross-platform reproducibility was improved and false positives reduced.

CONCLUSIONS

Optimized sample preparation combined with a curated metabolite database enables high-confidence, comprehensive profiling of broccoli florets phytochemicals. The resulting dataset provides a valuable reference for studies on genotype-environment interactions, nutritional quality, and functional bioactivity of cruciferous vegetables.