What does this strategy enable?
Suspect screening occupies the space between targeted analysis and fully non-target screening. Rather than requiring authentic standards for every analyte or searching every detected feature without prior hypotheses, the workflow begins with curated suspect lists and tests whether those candidates are supported by accurate mass, chromatographic peak quality, fragmentation evidence, and matrix-aware filtering. The 2024 workflow evaluated this approach across multiple human biofluids and explicitly measured false-positive and false-negative behavior.
Transferable workflow
Workflow
- 1
Build suspect lists
Assemble plausible parent xenobiotics and phase-II metabolites relevant to the exposure question and sample matrix.
- 2
Acquire UHPLC-HRMS/MS data
Analyze biofluids using high-resolution MS with fragmentation information suitable for candidate assessment.
- 3
Filter chromatographic features for peak quality
Apply automated peak-quality criteria to reduce poor chromatographic candidates before manual review.
- 4
Compare candidates with suspect and endogenous lists
Evaluate expected xenobiotic candidates while accounting for endogenous molecules that may create annotation interference.
- 5
Evaluate structural evidence
Combine accurate mass, isotope/adduct behavior, fragmentation evidence, and available reference information to annotate candidates.
- 6
Estimate identification errors
Assess false positives and false negatives using standards or spiked samples where available.
Connected resources
Tools used in this strategy
Strategy profile
Related objectives
Metabolite annotation, Unknown identification
Components
Sample preparation, Data acquisition, Preprocessing and feature extraction, Spectral search, Annotation or identification, Metadata analysis, Biological validation
Sample types
Urine, Saliva, Other biofluid
Biological context
Human
Organisms
Homo sapiens
Ion mobility
Ion mobility not required
Scope and considerations
Designed for
- Screening broad but hypothesis-informed sets of environmental or exogenous chemicals in human biofluids.
- Exposomics studies where fully targeted assays would be too narrow but unconstrained non-target identification is impractical.
Important requirements
- Curated suspect lists appropriate to the study question.
- High-resolution MS data and enough spectral/chromatographic evidence to evaluate candidates.
Considerations
- Suspect-list composition determines which exposure space is visible.
- Matrix interferences and endogenous molecules can create both false positives and false negatives.
Reusable resources
Publication
Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids. Chemosphere 351, 141221 (2024).