A Combined Method for Anionic, Cationic, and Zwitterionic PFAS using a Direct Injection UPLC™-MS/MS Method for Environmental Water Samples

Significance of the topic

The environmental occurrence of per- and polyfluoroalkyl substances (PFAS) has expanded beyond classical anionic species to include cationic and zwitterionic congeners, notably those associated with aqueous film forming foams (AFFF). Accurate, high-throughput quantitative methods that capture this chemical diversity are essential for regulatory compliance, environmental monitoring, and remediation decision-making. The ability to measure anionic, cationic and zwitterionic PFAS in a single, robust direct-injection UPLC-MS/MS workflow reduces analytical complexity and improves laboratory efficiency while maintaining sub-ng/L sensitivity required by EU and U.K. directives.

Objectives and overview of the study

The study aimed to extend a previously reported direct-injection UPLC-MS/MS PFAS method to include representative cationic and zwitterionic analytes alongside anionic PFAS in one chromatographic run. Key goals were to: establish a quantitative multiple reaction monitoring (MRM) method for 58 native PFAS plus 23 isotopically labeled internal standards (ILIS); optimize ionization and collision conditions to support fast polarity switching; validate performance across diverse environmental waters (surface, ground, treated, and wastewater); and demonstrate compliance with regulatory sensitivity and precision criteria.

Methodology

Sample preparation:

• Environmental water samples were screened, spiked with ILIS prior to processing, and subjected to a simple filtration/dilution workflow compatible with direct injection to minimize sample handling losses and adsorption artefacts.

Chromatography and acquisition:

• Direct injection onto ACQUITY UPLC system with ACQUITY Premier BEH C18 column; 50 µL injections; 20 min total run time with all analytes eluting within 16 min.
• Mobile phases contained 2 mM ammonium acetate; gradient optimized to achieve good peak shape and retention, while ensuring at least 12 points per peak (autodwell functionality used).

Mass spectrometry and ionization:

• Xevo TQ Absolute XR triple quadrupole mass spectrometer with UniSpray ionization operating in fast positive/negative polarity switching for simultaneous detection of cationic/zwitterionic (positive-favoring) and anionic (negative-favoring) PFAS.
• Nitrogen was used as collision gas (adjusted cell pressure and collision energies with conversion factor), chosen for sustainability (single gas supply) and improved transmission of low m/z product ions.
• Two MRMs optimized per native analyte; one dominant MRM per ILIS. Source and MS parameters were tuned to ensure sensitivity for the least responsive analytes.

Quantification and software:

• Solvent-based calibration (50:25:25 water:methanol:acetonitrile with 0.1% formic acid) spanning 0.12–240 ng/L with automatic purity/salt corrections applied in waters_connect MSQuan.
• ILIS added prior to preparation to correct matrix effects and enable solvent standard quantitation; composite ILIS strategies supported for analytes lacking exact analogues.

Used Instrumentation

• ACQUITY I-Class PLUS UPLC System with FTN/BSM and PFAS kit sample manager.
• ACQUITY Premier BEH C18 column (1.7 µm, 2.1 × 100 mm) and Atlantis Premier BEH C18 AX isolator column.
• Xevo TQ Absolute XR triple quadrupole mass spectrometer with UniSpray Ionization Source and StepWave XR ion transfer device.
• waters_connect for Quantitation suite (Advanced Method Editor, MSQuan, AME, Sample Sub, MS ToolKit).

Main results and discussion

Analytical performance:

• Sensitivity: LLOQs across the 58 PFAS ranged from 0.04 to 0.6 ng/L (surface water), meeting and often exceeding EU/U.K. requirements for drinking and environmental waters.
• Linearity: Excellent linearity with R2 ≥ 0.996 for all native PFAS and residual deviations within ±20% above LLOQ; at least three orders of dynamic range for every analyte.
• Precision and repeatability: Intra-batch repeatability at 2 ng/L (n=21) showed RSDs <14% for all analytes; retention-time RSDs <0.3% over 63 injections across three matrices; calculated concentration deviations <15% RSD.
• Apparent recovery: Measured recoveries in surface water (pre- vs post-spike) ranged from ~70% to 109%, with lower recoveries for more hydrophobic, long-chain PFAS consistent with adsorption/loss phenomena.
• Carryover and background: Use of FTN sample manager with HPS needle and a mixed solvent wash controlled carryover (no PFAS exceeded 30% of lowest calibration standard peak area); StepWave XR mitigated quadrupole contamination improving robustness during extended direct-injection campaigns.

Matrix effects and isomer separation:

• Matrix signal suppression of about 20% was observed for PFOA in different water types, underscoring the importance of ILIS correction; waters_connect features allow monitoring ILIS performance and applying composite ILIS where necessary.
• Chromatographic resolution allowed detection of branched versus linear isomers (e.g., PFOA), which may inform source attribution and fate studies.

Application to real wastewater:

• Analysis of a trade effluent detected 16 PFAS above reporting limits; several analytes exceeded 20 ng/L with L-PFHxS at 172 ng/L, L-PFOS at 134 ng/L, L-PFOA at 119 ng/L, and 6:2 FTAB (zwitterion) at ~82.2 ng/L.
• These findings validate the necessity of including cationic and zwitterionic PFAS in routine targeted monitoring, particularly near AFFF-impacted sites.

Benefits and practical applications

• Single-injection workflow captures diverse PFAS classes (anionic, cationic, zwitterionic), reducing analytical complexity and increasing laboratory throughput.
• Sub-ng/L sensitivity and broad linear dynamic range reduce need for reanalysis and dilution, supporting compliance testing across variable concentration ranges.
• Robust hardware (StepWave XR design) and optimized cleaning/wash protocols decrease maintenance downtime for high-volume direct-injection operations.
• Integrated software (waters_connect) streamlines method development, batch processing, QA/QC review and reporting, saving time and minimizing manual review.

Future trends and potential applications

• Expansion of targeted panels: The demonstrated scalability supports rapid inclusion of newly identified PFAS (including novel zwitterions and transformation products) as reference materials become available.
• Hybrid approaches: Combining direct injection screening with targeted solid-phase extraction or non-targeted HRMS for suspect screening could provide richer source attribution and transformation pathway information.
• Remediation monitoring: Sensitive quantification of cationic/zwitterionic PFAS will support evaluation of advanced treatment technologies (electrochemical oxidation, photocatalysis, adsorption, biodegradation) and their effectiveness for diverse PFAS chemistries.
• Regulatory alignment: Methods with fast polarity switching and ILIS-based quantitation will be increasingly important as regulatory bodies expand reporting lists and lower limits.

Conclusion

The combined direct-injection UPLC-MS/MS method successfully extends quantitative coverage to anionic, cationic, and zwitterionic PFAS within a single 20-minute run while achieving sub-ng/L LLOQs, excellent linearity, reproducibility and robustness across environmental water matrices. Use of UniSpray ionization, nitrogen collision gas, fast polarity switching, and isotopically labeled standards enabled reliable quantification and mitigated matrix and carryover challenges inherent to large-volume direct injection. The method supports regulatory monitoring and high-throughput laboratory workflows and can be scaled to include emerging PFAS as analytical needs evolve.

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