Detection and Characterization of PFAS Molecules Using Inclusion and Exclusion Features of Data Directed Analysis (DDA)

Importance of the Topic

Exposure to poly- and per-fluoroalkyl substances (PFAS) has been linked to adverse health outcomes, including cancer. Because PFAS accumulate in biological matrices at trace levels, their reliable detection and characterization require highly selective analytical approaches.

Study Objectives and Overview

This work demonstrates the integration of mass defect–based inclusion lists and matrix-derived exclusion lists within a data-directed acquisition (DDA) workflow on a cyclic ion mobility time-of-flight (ToF) mass spectrometer to enhance PFAS identification in complex backgrounds such as human serum.

Methodology

Liquid chromatography was performed on an ACQUITY Premier system equipped with PFAS-optimized kits and Atlantis Premier BEH C18 AX and HSS T3 C18 columns. Mobile phases consisted of methanol and water with ammonium acetate. A SELECT SERIES Cyclic IMS ToF mass spectrometer operated in negative-ion DDA mode isolated candidate PFAS precursors by quadrupole and generated MS/MS spectra with stepped collision energies. Data acquisition and processing used MassLynx v4.2 and waters_connect software, referencing an in-house PFAS library.

Main Results and Discussion

The use of a targeted inclusion list based on PFAS Kendrick mass defects (600 entries, 200–800 Da) allowed lowering the DDA trigger threshold and prioritizing PFAS precursors over abundant background ions. Simultaneously, an exclusion list (>9,000 matrix m/z values derived from blank serum) suppressed non-PFAS switches. This strategy enabled clear differentiation of isomeric PFOS fragments (e.g., perfluoro-5- and perfluoro-6-methylheptanesulfonic acids) by characteristic product ions (m/z 329.9) and improved detection of perfluorocarboxylic acids such as perfluorotetradecanoic acid (PFTreDA) with high mass accuracy (0.9 ppm RMS).

Practical Benefits

• Enhanced specificity for low-level PFAS in complex matrices
• Reduced unnecessary MS/MS events on background ions
• Improved confidence in isomer discrimination
• Streamlined workflow suitable for environmental and biomonitoring applications

Used Instrumentation

• Waters ACQUITY Premier UPLC with PFAS Kit
• Atlantis Premier BEH C18 AX and ACQUITY UPLC HSS T3 C18 columns
• SELECT SERIES Cyclic Ion Mobility ToF Mass Spectrometer
• MassLynx v4.2 and waters_connect software
• TIBCO Spotfire for data visualization

Future Trends and Potential Applications

Expansion of PFAS inclusion libraries and adoption of automated matrix mapping for exclusion lists will further improve throughput. Integration with high-resolution ion mobility separation, machine-learning–driven spectral interpretation, and cross-laboratory sharing of collision cross section databases can broaden applications in environmental monitoring, clinical biomonitoring, and e-waste exposure studies.

Conclusion

The combination of mass defect–guided inclusion and matrix-derived exclusion lists within a DDA framework on a cyclic IMS ToF platform effectively enhances PFAS detection and characterization at trace levels. This approach offers a robust, selective workflow for challenging analytical matrices.

Reference

1. McCullagh M, Lioupi A, Theodoridis G, Plumb R, Wilson I, Adams S. Enhanced Identification Confidence and Specificity for PFAS Analysis Using Cyclic Ion Mobility Mass Spectrometry Collision Cross Sections. Waters Application Note 720008536, 2025.
2. McCullagh M, Adams S, Tudor A, Goshawk J, Mortishire-Smith R, Megson D, Ansong Asante K, Bruce-Vanderpuije P. Combining Pattern Analysis and Cyclic Ion Mobility Mass Spectrometry to Research Per- and Polyfluoroalkyl Substances (PFAS) Exposure in E-waste Handlers. Waters Application Note 720008784, 2025.
3. McCullagh M, Marsden Edwards E, Adams S. Illustrating the Use of Cyclic Ion Mobility to Enhance Specificity for Branched-PFAS Isomer Analysis. Waters Application Note 720007823, 2025.
4. Sleno L. The Use of Mass Defect in Modern Mass Spectrometry. Journal of Mass Spectrometry. 2012;47(2):226–236.