Untargeted PFAS identification and targeted PFAS library screening workflows for groundwater analysis using a QTOF mass spectrometer

Importance of the Topic

Per- and polyfluoroalkyl substances (PFAS) represent a significant environmental and public health concern due to their persistence, bioaccumulation potential, and documented toxicity. Groundwater contamination by both regulated and emerging PFAS has driven the need for analytical methods capable of identifying hundreds to thousands of PFAS variants. Comprehensive characterization of novel PFAS supports risk assessment and informs regulatory decisions.

Objectives and Study Overview

This study presents complementary workflows for targeted library screening and untargeted identification of PFAS in groundwater using a quadrupole time-of-flight (QTOF) mass spectrometer. The primary goals were to generate a high-confidence library of 40 PFAS (EPA Method 1633), evaluate the performance of targeted screening in spiked groundwater samples, and demonstrate untargeted discovery via data-independent acquisition (DIA) MS/MS.

Methodology and Instrumentation

• Liquid Chromatography: Shimadzu Nexera LC with Shim-pack Scepter C18-120 analytical (2.1×100 mm, 3 µm) and delay (2.1×50 mm, 3 µm) columns. Mobile phases were 5 mM ammonium acetate in water (A) and methanol (B) at 0.25 mL/min, 45 °C, 1 µL injection. Gradient: 0–1 min 5–40 % B; 1–8 min 40–95 % B; 8.1–13 min 100 % B; 13.1–18 min 5 % B.
• Mass Spectrometry: Shimadzu LCMS-9030 QTOF in negative-ion electrospray mode (ESI). Full-scan MS (40–950 m/z), targeted MS/MS on selected precursor m/z, and DIA MS/MS with 5–55 V collision energy spread. Source conditions: nebulizing gas 3 L/min, drying gas 5 L/min, heating gas 15 L/min, DL 150 °C, desolvation 160 °C, heat block 250 °C, interface 100 °C.
• Data Processing: LabSolutions and LabSolutions Insight Explore software. Assign function enabled targeted library screening and untargeted formula/mass-based searches against ChemSpider and PubChem databases.

Main Results and Discussion

• A tailored library of 40 PFAS from EPA Method 1633 was successfully generated, covering a range of chemistries including short-chain (e.g., PFBA, PFBS), long-chain (e.g., PFOA, PFOS, PFUnA), halogenated species (e.g., 11Cl-PF3OUdS), and emerging analytes (e.g., HFPO-DA, ADONA, N-ethyl FOSA derivatives).
• Targeted screening of groundwater spiked with 15.6–390 ppb PFAS achieved confident identification of all compounds, with mass errors below 5 ppm and retention time shifts under 0.05 min.
• Untargeted DIA MS/MS enabled the detection of PFAS features not initially in the library by matching accurate mass and characteristic fragment ions, illustrating the workflow’s capability to discover novel PFAS.
• Comparison of MS/MS spectra between library standards and groundwater samples confirmed structural assignments, demonstrating high reproducibility and selectivity.

Benefits and Practical Applications

• Efficient and reliable detection of known and novel PFAS in environmental waters supports regulatory compliance and contamination mapping.
• Expandable targeted library allows rapid incorporation of newly identified PFAS for routine monitoring.
• Untargeted workflows serve as a screening tool for emerging PFAS, guiding method optimization and risk evaluation.

Future Trends and Opportunities

• Integration of advanced software with comprehensive public databases to automate unknown PFAS annotation.
• Development of inter-laboratory spectral libraries to standardize PFAS identification protocols.
• Incorporation of high-resolution ion mobility spectrometry to resolve PFAS isomers and coeluting species.
• Advancements in sample preparation and instrument sensitivity to achieve lower detection limits and high throughput for large-scale environmental monitoring.

Conclusion

The combined targeted library screening and untargeted DIA workflows on a QTOF platform provide a robust framework for comprehensive PFAS analysis in groundwater. The in-house library of 40 PFAS demonstrated excellent screening performance, while DIA strategies enabled the identification of non-target PFAS, strengthening analytical capabilities for environmental surveillance.

References

• EPA Method 1633: Per- and Polyfluoroalkyl Substances in Water by LC-MS/MS.
• Shimadzu Application Note: Untargeted PFAS identification and targeted PFAS library screening workflows for groundwater analysis using a QTOF mass spectrometer.