Analysis of PFAS in Water Samples Containing Insolubles Using Triple Quadrupole LC/MS/MS

Significance of the Topic

Per- and polyfluoroalkyl substances (PFAS) are persistent, bioaccumulative compounds widely used for their hydrophobic and thermal properties. Their environmental persistence and potential health impacts have driven stricter regulatory scrutiny and an increasing need for reliable quantitative methods across diverse water matrices. Standard PFAS methods for drinking water are not directly transferable to environmental or agricultural waters containing significant suspended solids; therefore, validated procedures that accommodate insolubles without compromising recovery or damaging SPE cartridges are essential for accurate monitoring and regulatory compliance.

Study Objectives and Overview

The work demonstrates an analytical workflow, based on the NARO (National Agriculture and Food Research Organization) manual, for quantifying 30 PFAS in water samples containing insoluble suspended solids using triple-quadrupole LC/MS/MS (Shimadzu LCMS-8060RX). Goals were to: (1) apply an extraction and clean-up approach compatible with small-volume samples that include milligram levels of suspended solids, (2) achieve simultaneous quantification of 30 PFAS with good linearity and sensitivity, and (3) evaluate recovery, repeatability, and internal standard performance in both procedure blanks and real agricultural water samples.

Methodology

• Sample handling: 40 mL water aliquots in 50 mL polypropylene tubes. An isotopically labeled surrogate mix (100 µL, 10 ng/mL) was added, samples vortexed and centrifuged (3,000 rpm, 10 min) to separate supernatant and suspended solids.
• Solid fraction extraction: Suspended solids were extracted three times with 5 mL of 0.5% ammonia in methanol (mixing and horizontal shaking ~30 min each), centrifuged, and pooled with the initial supernatant to yield ~55 mL final volume. Acetic acid (1 mL) was added to the pooled extract.
• SPE clean-up: Anion-exchange SPE (InertSep WAX FF, 150 mg/6 mL) was used. Conditioning sequence included 0.1% ammonia/methanol, methanol, and ultrapure water followed by acetate buffer. The sample was loaded, washed, and eluted in two fractions: neutral substanced fraction (methanol) and anionic fraction (0.1% ammonia/methanol). Fractions were combined as appropriate.
• Concentration and reconstitution: Final extracts were evaporated under N2 at 35 °C to <1 mL and reconstituted to 1 mL with methanol prior to LC/MS/MS analysis.

Instrumentation Used

• UHPLC: Nexera X3 system with Shim-pack Scepter C18-120 analytical column (100 × 2.1 mm, 1.9 µm) and Shim-pack Scepter guard/EXF holder. A PFAS-specific delay column was employed to mitigate background contamination.
• Mass spectrometer: Shimadzu LCMS-8060RX triple quadrupole, electrospray ionization (negative mode).
• Chromatographic conditions: Mobile phases A = 2 mmol/L ammonium acetate in water, B = methanol; gradient from 5% B to 100% B with a flow-rate ramp (0.3 → 0.6 → 0.3 mL/min) and column temperature 40 °C. Injection volume 5 µL.
• MS source: ESI negative, interface temp 250 °C, heat block 300 °C, DL temp 200 °C, interface voltage −1.0 kV, multiple MRM transitions optimized for 30 target PFAS and corresponding 13C or deuterated internal standards.

Target Compounds and Calibration

The method targets 30 PFAS spanning perfluoroalkyl sulfonates and carboxylates, fluoroalkyl sulfonamides/precursors (FOSA, Me/Et-FOSA, Me/Et-FOSAA), fluorotelomer sulfonates (6:2 FTSA, 8:2 FTSA), fluorotelomer phosphate/diPAPs, HFPO-DA (GenX), DONA, and other long-chain homologues up to C20 analogues. Calibration standards (mixed PFAS and internal standard mixes) were prepared in methanol across multiple levels (typical solution ranges 0.002–10 ng/mL depending on analyte). Most calibration curves exhibited excellent linearity (R ≈ 0.998–0.9999) using linear fit with 1/C weighting and non-forced zero.

Main Results and Discussion

• MRM chromatograms for a 0.2 ng/mL mixed standard set demonstrated resolved peaks for all monitored PFAS within a run time of 28 min using the described gradient.
• Procedure blank and spike recovery (spike to 25 ng/L in 40 mL water) results: Recoveries for 29 of 30 target analytes ranged from 91 to 107%. One compound, 6:2 FTSA, showed elevated and variable recovery (up to 141%), consistent with known contamination and variability for this analyte. Procedural blank concentrations were generally low.
• Agricultural field water (rice field water) containing several mg of suspended solids per 40 mL was analyzed in triplicate following the same pretreatment. Quantitative results showed intraday repeatability (%RSD, n = 3) below 6% for all compounds except 6:2 FTSA, confirming robust repeatability in a challenging matrix.
• Internal standard performance: Most 13C-labeled standards had recoveries in the acceptable range (≈77–115%). Notable exceptions were lower recoveries for some neutral-labeled surrogates (d3-N-MeFOSA and d5-N-EtFOSA, ~49–73%) and an elevated recovery for 13C4-8:2 diPAP (~146%), indicating variable behavior among classes and the importance of careful internal standard selection and quality control.

Practical Benefits and Applications

• This protocol allows reliable PFAS quantification in small-volume environmental and agricultural water samples that contain suspended solids without the need for filtration that could reduce analyte recovery.
• The combined centrifugation-plus-solid-extract approach prevents SPE cartridge clogging while enabling extraction of PFAS that partition into the particulate fraction, improving method comprehensiveness for mixed-phase samples.
• High calibration linearity and generally acceptable spike recoveries support application for environmental monitoring, regulatory surveillance, and research studies targeting PFAS contamination in waters impacted by agricultural runoff, surface-water mixing, or wastewater inputs.

Future Trends and Potential Applications

• Enhanced contamination control: Additional laboratory measures and ultra-clean workflows are needed to minimize variable contamination, particularly for susceptible compounds such as 6:2 FTSA.
• Improved surrogate coverage: Expanding and optimizing isotope-labeled internal standards for neutral PFAS and precursors will improve quantitation accuracy across compound classes.
• Integration with high-resolution and non-target screening: Complementary HRMS workflows would allow identification of emerging PFAS and transformation products not covered by targeted MRM panels.
• Automation and miniaturization: Automated SPE platforms and microextraction approaches could increase throughput and reduce solvent use while preserving performance for particulate-rich waters.
• Method harmonization: Continued alignment of laboratory procedures with evolving regulatory manuals and inter-laboratory validation will support standardized monitoring networks and consistent reporting.

Conclusion

Following the NARO manual, the LCMS-8060RX triple-quadrupole platform and the described sample preparation can reliably quantify 30 PFAS in water samples containing insolubles. The protocol achieves excellent calibration linearity and acceptable recoveries for most analytes, with robust repeatability in agricultural water matrices. Attention to blank control and internal standard selection is essential, particularly for analytes prone to contamination or differential extraction behavior (e.g., 6:2 FTSA and certain neutral PFAS surrogates). Practitioners should consult the latest version of the NARO manual and implement strict laboratory controls to maintain data quality.

Reference

• National Agriculture and Food Research Organization. Determination of Perfluoroalkyl and Polyfluoroalkyl Substances (PFAS) in Water — From Sample Collection to Measurement — Version 1.0.

Acknowledgments

This work was performed under the research program Regulatory research projects for food safety, animal health and plant protection (JPJ008617.23812803), funded by the Ministry of Agriculture, Forestry and Fisheries of Japan. The analytical workflow and instrumental performance were demonstrated using Shimadzu Nexera X3 and LCMS-8060RX systems with Shim-pack Scepter C18 columns and InertSep WAX FF SPE cartridges.