Beyond compound‑specific methods: Group‑specific UHPLC-MS/MS enables rapid detection and quantitation of all...

Glucosinolates are widely consumed specialized metabolites predominantly found in the Brassicaceae family. Existing analytical methods typically target only specific subsets of glucosinolates and therefore lack the versatility required for comprehensive glucosinolate screening. Hence, there is a gap in analytical methods targeting glucosinolates that can detect any glucosinolate within a given matrix. In this study, targeted compound group‑specific multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods were developed and validated for comprehensive qualitative and quantitative profiling of glucosinolates. Among all glucosinolate group-specific fragment ions evaluated, five (m/z 96, 97, 241, 259, and 275) were selected for product ion optimization and method validation. Of the fifteen transitions selected for method validation, four were retained in the final MRM method based on sensitivity and selectivity. Additionally, the performances of the group-specific methods were compared against simultaneously created compound-specific MRM methods. Although the more traditional compound‑specific methods offered slightly improved sensitivity, they were limited to detect only individual glucosinolates. While detecting whole glucosinolate groups, the group‑specific MRM methods showed lowest limits of detection (LOD) ranging from 11.5 to 87.3 ng/mL, and the upper limits of quantitation (ULOQ) from 2.5 to 55 µg/mL. High‑resolution group-specific PRM methods enabled accurate glucosinolate characterization and yielded LODs for the best precursor ion m/z 259 between 44.1 and 473.6 ng/mL, with ULOQs ranging from 8 to 50 µg/mL. Application of the methods demonstrated their capability to detect 51 distinct glucosinolates across 19 plant species.