Dual-Ionization SPME-GC–HRMS Metabolomic Profiling of Broccoli Volatiles for the Construction of a Broccoli Metabolic Database

Volatile organic compounds (VOCs) play critical roles in broccoli’s sensory attributes, defense mechanisms, and ecological interactions, yet comprehensive profiling of its volatilome remains limited. This study aimed to construct a robust and inclusive volatile metabolite database for broccoli using advanced analytical techniques. A pooled sample comprising florets from 191 cultivars was prepared to capture broad chemical diversity and analyzed using solid-phase microextraction–gas chromatography–high-resolution mass spectrometry (SPME-GC-HRMS) under dual ionization modes: electron ionization (EI) and chemical ionization (CI). A total of 206 VOCs spanning nine chemical classes were detected, with 37 compounds further confirmed through synchronized CI analysis. To validate the database, broccoli florets from seven distinct cultivars were analyzed using the same workflow. Of the 206 compounds, 187 (90.78%) were detected in at least one cultivar, while 38 were consistently found across all samples, indicating a conserved core volatilome. Principal component analysis revealed distinct VOC profiles among cultivars, and freeze-dried samples were found suitable for reproducible large-scale analysis. This study demonstrates that a pooled-sample strategy coupled with dual-ionization GC-HRMS provides comprehensive and reliable VOC coverage. The resulting database offers a valuable resource for metabolomics studies in Brassica, with applications in cultivar differentiation, flavor research, and environmental response profiling.