A confidence-tiered LC-MS workflow for microcystin congener profiling via targeted quantification, database-assisted...

Microcystins (MCs) are structurally diverse hepatotoxic cyanotoxins of environmental and public-health concern, yet congener-resolved monitoring remains constrained by limited reference standards. This study developed a confidence-tiered ultra-high-performance liquid chromatography mass-spectrometry (UHPLC-MS) workflow to extend reported MC congener profiling while preserving quantitative reliability under standard-limited conditions. The workflow integrates targeted UPLC-MS/MS quantification of standard-covered congeners with database-assisted non-targeted screening of reported congeners beyond the targeted panel using UPLC-high-resolution mass-spectrometry (UHPLC-HRMS). A targeted method was established for 12 representative standard-covered MC congeners, and UHPLC-HRMS characterization of these standards was used to derive recurrent fragmentation rules for the conserved motif at positions 5∼7 and related substructures broadly retained among reported MCs. These rules were used to construct a structure-informed database covering 299 reported MC congeners, supporting precursor- and fragment-based matching. Workflow outputs were classified into three confidence levels: standard-confirmed quantitative results (Level 1), uniquely matched reported congeners lacking standards with semi-quantitative estimates (Level 2), and unresolved multi-match features retained as tentative candidate groups (Level 3). Application to a prepared surface-water sample containing a toxin-producing M. aeruginosa biomass extract generated a confidence-tiered profile comprising 10, 5, and 3 outputs respectively for Level 1, Level 2, and Level 3. MC-WR was the dominant standard-supported congener, reaching 1.46 × 10⁶ µg/kg. For MC-like features unassigned by the primary workflow, a virtual-candidate strategy based on 282,356 non-reported structures generated from reported-MC residue patterns provided prioritized exploratory hypotheses. The confidence-tiered workflow integrates quantification, non-targeted screening, and virtual-candidate strategy to expand congener-resolved MC profiling under limited standards.