Anticipating emerging regulations: Direct injection LC-MS/MS quantification of C1–C4 ultrashort-chain PFAS in drinking water using the TSQ Altis Plus EFOX Edition

Importance of the topic

The increasing regulatory attention to per- and polyfluoroalkyl substances (PFAS) has shifted focus toward ultrashort-chain PFAS (USC-PFAS, C1–C4). These compounds are highly polar, mobile in water, difficult to remove in treatment, and may occur at ultra-trace concentrations in drinking water. Reliable, selective, and sensitive analytical methods are therefore essential for routine monitoring and for meeting emerging regulatory requirements.

Objectives and overview of the study

This application note describes a forward-looking direct-injection LC-MS/MS workflow for quantifying USC-PFAS (C1–C4) in drinking water. The method pairs HILIC chromatography (Hypersil GOLD HILIC column) with a TSQ Altis Plus EFOX Edition triple quadrupole mass spectrometer operated in SRM and dual-CID (DSRM, MS3-like) modes. The method aims to overcome retention and sensitivity challenges for highly polar PFAS, provide low ng/L quantitation where feasible, and be compatible with routine laboratory workflows and anticipated regulatory expansion.

Methodology

Key methodological elements:

• Sample preparation: direct injection after simple dilution (1:1 v/v water:acetonitrile) to reduce elution strength, control matrix effects, and enable robust direct injection.
• Chromatography: Hypersil GOLD HILIC (100 x 2.1 mm, 1.9 µm) with a 50 x 2.1 mm delay HILIC column; column temperature 40 °C; injection volume 5 µL; flow 400 µL/min; run time 15 min. Mobile phase A: 20 mM ammonium formate in water; Mobile phase B: acetonitrile 95% / isopropanol 5% (v/v). Short gradient and fast re-equilibration optimized for throughput and retention of highly polar acids (notably TFA).
• Mass spectrometry: TSQ Altis Plus EFOX Edition triple quadrupole in negative HESI; SRM for quantitation plus dual-CID (DSRM) for MS3-like confirmation (sequential S-CID and ARC-CID). This dual-CID approach increases confirmation ion availability and selectivity (example: TFSI generates 280→147 quant ion and DSRM 280→147→78 confirmation ion).
• Calibration and internal standards: isotope dilution with multiple 13C-labeled internal standards (e.g., 13C2-TFA, 13C3-PFPrA, 13C4-PFBA, 13C3-PFBS) to compensate for matrix effects and instrument variability. Calibration ranges typically 5–5,000 ng/L (TFA calibrated from 0.5–50 µg/L due to background), using weighted quadratic fits with offset.
• Contamination control: PFAS-free LC-MS solvents and polypropylene labware; elimination of PTFE components where possible; system and solvent blanks in every batch; LOQs set according to laboratory background levels.

Instrumentation

Main hardware and software used:

• Vanquish Duo UHPLC platform (Vanquish Flex Binary UHPLC Pump, Dual Split Sampler, Column Compartment)
• Hypersil GOLD HILIC analytical and delay columns (100 x 2.1 mm and 50 x 2.1 mm, 1.9 µm)
• TSQ Altis Plus EFOX Edition Triple Quadrupole Mass Spectrometer (negative HESI)
• Chromeleon CDS software for instrument control, acquisition, and data processing

Main results and discussion

Chromatography and retention:

• HILIC separation produced reproducible retention and sharp peaks for USC-PFAS, including clear separation of TFA from the solvent front, minimizing matrix coelution.

Sensitivity and linearity:

• Method LOQs (solvent) generally ranged from 5 to 100 ng/L for most analytes; TFA LOQ limited to 500 ng/L (solvent) and 1,000 ng/L as method quantification limit (MQL) due to laboratory background contamination.
• Calibration performance: R² values >0.990 for all analytes across calibration ranges; validation criteria met (R² >0.990 and relative deviations within acceptance criteria).

DSRM (dual-CID) performance and selectivity:

• Dual-CID transitions provided MS3-like confirmation ions that are more selective/intense than conventional SRM confirmation ions, improving confidence at trace levels (illustrated by TFSI and applied to PFBSAm and PFBA).

Matrix effects and robustness:

• A notable “salt plug” from inorganic matrix components (Na+, Ca2+, Mg2+, HCO3−, Cl−) produced ion suppression around specific retention windows. Isotope-labeled standards corrected most suppression effects, although some IS (e.g., 13C3-PFPrA) exhibited ~80% attenuation in real samples.
• Long-sequence robustness testing (many injections, fortified matrices) showed stable retention times, acceptable RSDs for calculated concentrations (<20% under repeatability testing), minimal carryover, and sustained column performance for routine use.

Real sample analysis:

• Six drinking water samples (two bottled mineral waters, three tap waters, one water fountain) were analyzed. Most USC-PFAS were below MQL or non-detectable. PFBA and PFPrA occurred at tens of ng/L in some samples, but only one bottled water exceeded the PFPrA MQL. TFA was detected in multiple tap water samples at concentrations above the MQL (values reported ~1,496–2,351 ng/L in some taps).
• Spike-recovery experiments across three levels demonstrated recoveries generally within ±20–30% for most compounds; RSDs for recoveries were typically below 30%, indicating acceptable accuracy and precision for routine monitoring.

Benefits and practical applications

This method offers:

• Robust direct-injection workflow compatible with routine drinking-water monitoring, minimizing sample prep time and complexity.
• Improved chromatographic retention and separation of highly polar USC-PFAS via HILIC, overcoming limitations of reversed-phase approaches.
• Enhanced selectivity through SRM complemented by DSRM (dual-CID), providing MS3-like confirmation ions to increase confidence at ultra-trace levels.
• Isotope-dilution correction to mitigate matrix effects and instrument variance.
• Scalability and integration potential using Vanquish Duo and EFOX configuration to support concurrent USC-PFAS and conventional PFAS analyses on a single high-performance platform.

Future trends and potential uses

Anticipated developments and opportunities:

• Regulatory expansion: as limits for USC-PFAS tighten, laboratories will need lower environmental backgrounds (cleaner labs and consumables) and improved LOQs through contamination control and optimized workflows.
• Method refinements: further lowering LOQs via enhanced source cleanliness, dedicated PFAS-free infrastructure, or alternative sample prep (e.g., SPE variants) while balancing throughput.
• Broader analyte panels: inclusion of additional ultrashort precursors and transformation products as standards become available.
• Integration with treatment assessment: coupling sensitive monitoring with studies on removal efficiencies and formation pathways during water treatment.
• High-resolution and orthogonal confirmation strategies: combining DSRM with HRMS or complementary ionization/fragmentation approaches to strengthen non-target screening and identification.

Conclusion

The presented direct-injection HILIC LC-MS/MS method using the TSQ Altis Plus EFOX Edition with dual-CID capabilities provides a practical, selective, and robust approach for quantifying C1–C4 USC-PFAS in drinking water. The workflow balances sensitivity, throughput, and routine-lab compatibility while delivering improved retention for highly polar PFAS and MS3-like confirmation power. Laboratories adopting this approach should emphasize contamination control to achieve the best LOQs and be prepared to adapt as regulatory limits evolve.

References

1. Jian, J. M.; et al. A short review on human exposure to and tissue distribution of per- and polyfluoroalkyl substances (PFASs). Science of The Total Environment 2018, 636, 1058–1069.
2. Chow, S. J.; et al. Detection of ultrashort-chain and other per- and polyfluoroalkyl substances (PFAS) in U.S. bottled water. Water Research 2021, 201, 117292.
3. European Parliament and Council of the European Union. Directive (EU) 2020/2184 of 16 December 2020 on the quality of water intended for human consumption.
4. U.S. Environmental Protection Agency (EPA). Drinking Water Research Methods: Per- and Polyfluoroalkyl Substances (PFAS).
5. Neuwald, I. J.; et al. Ultra-Short-Chain PFASs in the Sources of German Drinking Water: Prevalent, Overlooked, Difficult to Remove, and Unregulated. Environmental Science & Technology 2022, 56, 6380–6390.
6. Thermo Fisher Scientific. Application Brief 003941: Out-of-the-box workflow for PFAS quantitation using a targeted approach with the TSQ Altis Plus mass spectrometer.
7. Thermo Fisher Scientific. Application Note 002902: Direct injection of drinking water for the analysis of 54 PFAS compounds by LC-MS/MS aligned with current and evolving global regulations.
8. Taniyasu, S.; et al. Analysis of trifluoroacetic acid and other short-chain perfluorinated acids (C2–C4) in precipitation by liquid chromatography–tandem mass spectrometry: Comparison to patterns of long-chain perfluorinated acids (C5–C18). Analytica Chimica Acta 2008, 619, 221–230.
9. Liang, S.-H.; et al. Analysis of ultrashort-chain and short-chain (C1 to C4) per- and polyfluorinated substances in potable and non-potable waters. Journal of Chromatography Open 2023, 4, 100098.