Enhanced performance for FOSAs and FOSEs analysis with an ultra-sensitive PFAS-multiclass method in environmental samples

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are increasingly regulated and monitored due to their persistence, mobility and health concerns. Laboratories require robust, sensitive multiclass analytical workflows to quantify a broad spectrum of PFAS across environmental matrices (drinking water, wastewater, tissues). Methods that lower detection limits while preserving accuracy and throughput are critical to meet evolving regulatory limits (including emerging EU limits and EPA Method 1633A) and to reliably measure historically difficult PFAS classes such as FOSAs and FOSEs.

Study objectives and overview

The study validated two complementary LC–MS/MS workflows on the Shimadzu LCMS-8065XE platform for ultra-sensitive multiclass PFAS analysis: a Direct Injection approach optimized for maximal sensitivity in aqueous samples (29 PFAS targets) and an Extraction-based method aligned with EPA 1633A for comprehensive tissue and extracted-matrix analysis (40 PFAS targets). Emphasis was placed on method linearity, limits of detection/quantitation, recovery, reproducibility and robustness during extended operation. Particular attention was paid to compounds that often underperform in multiclass methods (FOSAs, FOSEs and other late-eluting species).

Methodology

The work implemented two laboratory workflows:

• Direct Injection: Samples prepared in 50/50 H2O/MeOH with co-solvation; internal standards added (final IS levels 10–40 ng/L depending on compound). Calibration range: 1–100 ng/L (7-point curves). Seven replicate spikes at 1 ng/L were used to calculate method detection limits (MDLs).
• Extraction-based (EPA 1633A-oriented): Solid-phase extraction or solvent extraction of tissues and complex matrices; calibration prepared in methanol with 4% water, 1% NH4OH and 0.625% acetic acid. Calibration ranges varied by analyte (native PFAS 2.5–62,400 ng/L; extracted internal standards 1–20 µg/L; non-extracted IS 1–4 µg/L). Method performance was monitored by low-level continuing calibration verification (CCV) and QA/QC per EPA 1633A.

Key validation metrics reported include linearity (R2 > 0.995 for extraction-based curves), calibration-point accuracy, relative standard error (RSE 3–17% for extraction-based), recoveries and reproducibility across matrices.

Instrumentation used

The analytical platform and key components used for method development and validation were:

• Mass spectrometer: Shimadzu LCMS-8065XE equipped with a new StreamFocus ESI probe and UFsweeper IV collision cell to enhance ionization efficiency and reduce background/contamination effects.
• Liquid chromatography: Shimadzu Nexera series UHPLC (Nexera X3/Nexera 40 referenced), with a dedicated delay column installed between mixer and autosampler to separate PFAS present in the HPLC system from sample-derived PFAS.
• Columns: Shimadzu Scepter C18-120 delay and analytical columns (examples: 2.1×100 mm 3 µm delay; 2.1×50 mm 1.9 µm analytical).
• Typical LC/MS settings summarized: nebulizing gas ~1.1 L/min, heating gas 15 L/min, drying gas 5 L/min; interface temperatures ~200–225 °C; DL and heat block 200–250 °C; interface voltage -0.2 to -0.5 kV; injection volumes 5 µL (direct) and 20 µL (extraction); flow rates 0.25–0.3 mL/min; mobile phases based on 2 mM ammonium acetate in water and methanol or acetonitrile.

Key results and discussion

Analytical figures of merit and notable outcomes from the validation:

• Sensitivity and linearity: Direct Injection calibration (1–100 ng/L) showed good linearity across 29 targets; extraction-based curves yielded R2 > 0.995 across 7 calibration points (compound-dependent concentration ranges up to 62,400 ng/L).
• Calibration accuracy: Direct Injection calibration points fell within 80–120% for PFOA and PFOS and within 70–130% for other targets. Extraction-based calibration point accuracies were also within 70–130% with RSE between 3% and 17%, meeting EPA 1633A QA/QC criteria.
• Method detection limits (MDLs): For Direct Injection, MDLs were below 1 ng/L for the target list without sample preconcentration, supporting compliance with stringent European and Water Framework Directive candidate limits.
• Recovery and reproducibility: Drinking water spiked at 4 ng/L showed recoveries generally in the 80–120% range and reproducibility <20% (n=7).
• Tissue LOQs and cross-class performance: Extraction-based LOQs in tissue ranged from ~0.003 ng/g to 0.625 ng/g. The new ESI source and collision cell produced improved sensitivity across PFAS classes, including historically poor performers (FOSAs, FOSEs, FTSs), with LOQs lower than or comparable to EPA 1633A reported minima.
• Chromatography and throughput: The 40-target PFAS panel was separated in a 10-minute run for the extraction method; cholic acids eluted distinctly earlier than both branched and linear PFOS by ~1.5 minutes, meeting EPA 1633A separation requirements. Measurement times: 10 min (direct) and 18 min (extraction) reported in method variants.
• Robustness and high-throughput operation: Over 930 injections of extracted tissue samples were performed with continual CCV monitoring (73 CCVs); all CCV accuracies remained within 70–130%, demonstrating stable performance over extended sequences without instrument cleaning or maintenance.

Benefits and practical applications

The validated approaches deliver several practical advantages:

• Direct Injection enables ultra-sensitive monitoring of aqueous samples without time-consuming preconcentration, simplifying workflows for routine drinking water surveillance.
• Extraction-based method aligns with EPA 1633A but extends sensitivity (CCVs at concentrations substantially lower than EPA LLOQ), permitting trace-level tissue analysis and low-ng/g LOQs.
• Improved ESI source and collision cell produce more even sensitivity across diverse PFAS chemistries, reducing class-specific performance gaps (notably for FOSAs/FOSEs) in multiclass methods.
• High throughput capability and long-run stability (hundreds of injections without maintenance) support regulatory testing labs and large monitoring campaigns.

Future trends and potential uses

Anticipated directions and applications building on this work include:

• Expansion of target lists and integration with high-resolution MS for suspect and non-target PFAS screening to capture emerging derivatives and transformation products.
• Automation and further simplification of sample preparation for higher throughput and lower per-sample labor.
• Interlaboratory standardization and round-robin studies to harmonize LOQs and QA/QC across laboratories and methods as regulation tightens.
• Continued hardware and ion-source evolution to further close sensitivity gaps for specific PFAS subclasses and to lower routine environmental regulatory limits.
• Application to broader matrices (food, consumer products) and coupling with bioaccumulation and risk-assessment workflows.

Conclusion

This study demonstrates that combining a modern ESI source (StreamFocus), an advanced collision cell (UFsweeper IV) and tailored LC conditions on the LCMS-8065XE platform enables highly sensitive, accurate and robust multiclass PFAS analysis across aqueous and extracted matrices. The two validated workflows—Direct Injection for drinking water and Extraction-based for tissue/complex matrices—achieve low ng/L MDLs, low ng/g LOQs in tissue, good recovery/reproducibility and exceptional long-run stability, while improving detection of historically difficult PFAS classes. These capabilities support regulatory compliance, routine monitoring and high-throughput laboratory operations.

References

• An Ultra-high Sensitivity Analysis of 29 PFAS Compounds in Drinking Water by Direct Injection.
• Breakthrough Sensitivity and Robustness for PFAS Analysis for EPA Method 1633A.

Acknowledgement: extracted solid samples were provided by CEM Corporation.