Identification of Anthropogenic Compounds in Stream Waters Using Non- target Strategies by HRMS

Significance of the topic

The presence of anthropogenic compounds in surface waters from wastewater effluents poses risks to ecosystems and human health. Non-target analysis with high-resolution mass spectrometry (HRMS) can uncover a wide range of emerging contaminants beyond traditional target lists, guiding water quality assessment and management.

Objectives and Study Overview

This study compared stream water collected along the Big Thompson River over five years at pristine upstream and urban-impacted downstream sites near Rocky Mountain National Park and Estes Park. The aim was to detect and identify as many anthropogenic compounds as possible using non-target HRMS and complementary data analysis strategies.

Methodology

• Sample collection from six locations along the river, representing control and impacted sites.
• Solid-phase extraction of 100 mL water samples on Oasis HLB cartridges via automated SPE.
• Elution with methanol, nitrogen drying to 0.5 mL, and 20 µL injection into LC/Q-TOF MS.
• Reverse-phase chromatography on a C8 column; combined All Ions MS/MS and iterative data-dependent MS/MS acquisition in positive electrospray.
• Data processing with Agilent MassHunter Explorer for feature extraction and ChemVista Library Manager for spectral library management.
• LOESS normalization and statistical comparison to identify compounds elevated at downstream sites (p < 0.05, ≥10-fold increase).

Instrumentation

• Agilent 1290 Infinity II liquid chromatograph
• Agilent 6546 LC/Q-TOF mass spectrometer
• Gilson GX-271 ASPEC automated SPE system

Main Results and Discussion

• A total of 5 487 features were detected across all samples.
• PCA revealed clear separation between upstream and downstream sites after normalization.
• 294 compounds were significantly elevated downstream by at least ten-fold.
• 27 compounds were confirmed using retention time and in-house MS/MS libraries.
• 22 additional substances were identified with the 2023 NIST MS/MS library.
• SIRIUS and CSI:FingerID supported structure confirmation for many NIST hits and proposed structures for compounds lacking library spectra.
• Confidence categories: ~20% identified at >95% confidence by combined strategies; 75% had proposed structures requiring manual validation; 5% yielded only molecular formulas.

Benefits and Practical Applications of the Method

Non-target HRMS strategies expand the scope of environmental monitoring by revealing known and unexpected contaminants. The combined use of spectral libraries and in silico tools enhances identification confidence, supporting regulatory decision-making, risk assessment, and pollution source tracing.

Future Trends and Opportunities

• Expansion and curation of high-quality tandem MS libraries for environmental compounds.
• Integration of machine learning to accelerate feature annotation and reduce false positives.
• Real-time data processing workflows for near-instant detection during field sampling.
• Coupling non-target HRMS with complementary techniques, such as ion mobility or nuclear magnetic resonance, for orthogonal confirmation.

Conclusion

This study demonstrates a robust non-target HRMS workflow to screen and identify anthropogenic pollutants in stream waters, highlighting the value of multi-tiered data analysis. Continued development of databases and computational tools will further improve detection confidence and expand applications in environmental chemistry.

Reference

1. Dührkop K., Fleischauer M., Ludwig M., Aksenov A.A., Melnik A.V., Meusel M., Dorrestein P.C., Rousu J., Böcker S. SIRIUS4: a rapid tool for turning tandem mass spectra into metabolite structure information. Nat Methods. 16, 2019.
2. Dührkop K., Shen H., Meusel M., Rousu J., Böcker S. Searching molecular structure databases with tandem mass spectra using CSI:FingerID. Proc Natl Acad Sci U S A. 112, 2015.