Out-of-the-box workflow for PFAS quantitation using a fullscan high-resolution approach with the Orbitrap Exploris EFOX Mass Detector

Out-of-the-box workflow for PFAS quantitation using a full-scan high-resolution approach with the Orbitrap Exploris EFOX Mass Detector — Summary

Significance of the topic

PFAS (per- and polyfluoroalkyl substances) are persistent, bioaccumulative synthetic chemicals of high environmental and public‑health concern. Regulatory frameworks in Europe and elsewhere are evolving rapidly, mandating sensitive, reliable analytical workflows for drinking water, surface water, groundwater and wastewater. High-resolution mass spectrometry (HRMS) workflows that combine targeted quantitation, suspect screening and retrospective data analysis offer laboratories flexibility to respond to changing target lists and stricter limits while maintaining trace-level quantitation and robust confirmation criteria.

Goals and overview of the study

The objective was to develop and qualify a ready-to-use full‑scan HRMS workflow, based on the Thermo Scientific Orbitrap Exploris EFOX Mass Detector, for quantitative analysis of a broad panel of 61 PFAS in methanolic extracts of water samples. The method aims to:

• Provide quantitative performance compatible with current European regulatory thresholds (e.g., drinking water criteria of 0.1 µg/L for a sum of 20 PFAS and 0.5 µg/L for total PFAS) and anticipated surface‑water standards.
• Offer orthogonal confirmation (dual‑column retention times and accurate mass) and the ability for suspect/retrospective screening from full‑scan data.
• Deliver sensitivity compatible with common concentration workflows (SPE, DLLME) and robust performance across matrices and extended sequences.

Methodology and experimental approach

The analytical strategy uses LC coupled to full‑scan HRMS (Orbitrap Exploris EFOX) operating at 60,000 resolving power with negative HESI source. Key method elements:

• Chromatography: Two orthogonal reversed‑phase columns (C8 and C18 Hypersil GOLD columns, 100 × 2.1 mm and 50 × 2.1 mm formats in the hardware bundle) are used in a dual‑path arrangement. The workflow performs two separations per sample within a single run and single data file to provide two retention times for orthogonal confirmation.
• Mobile phases: Water and methanol both containing 0.1 mM ammonium fluoride.
• Acquisition: Full‑scan accurate mass (m/z 70–1,000) with in‑source fragmentation option for additional fragment confirmation; scan resolution 60,000; polarity negative; injection volume 10 µL; total runtime ~14.5 min.
• Calibration and standards: Solvent calibration standards prepared in methanol from 25–5,000 ng/L with a mix of 22 isotopically labeled internal standards at 500 ng/L to correct for extraction and matrix effects. Spiking solutions prepared at 1 and 10 ng/mL for method qualification.
• Sample preparation: Compatible with common concentration/extraction approaches including SPE and DLLME; method qualification used neat solvent and SPE/DLLME extracts to assess matrix behavior.
• Quality strategy: Regular QC injections (QC at LOQ every 10 matrix injections), multiple calibration sets from independent days and operators, and stability/robustness testing across 40 matrix extracts over ~17 hours.

Instrumentation used

The evaluated configuration is an integrated Thermo Scientific workflow including:

• Orbitrap Exploris EFOX Mass Detector (HRMS)
• Vanquish Flex Binary UHPLC pumps (two independent pump paths)
• Vanquish Duo Autosampler with dual split sampler
• Vanquish Flex column compartment
• Hypersil GOLD columns: 50 × 2.1 mm and 100 × 2.1 mm, 1.9 µm (C18 and C8 chemistries)
• Chromeleon 7.3.2 CDS with supplied methods and data processing templates for guided review and reporting

Main results and discussion

Performance characteristics from method qualification:

• Sensitivity (solvent LOQ): Most compounds achieved solvent LOQs between 50 and 250 ng/L for a 10 µL injection; a small fraction required higher LOQs up to 500 ng/L. These solvent LOQs are compatible with typical SPE concentration factors (e.g., 100–500×), producing matrix LOQs on the order of 0.1–1 ng/L with 500× concentration.
• Linearity: Calibration curves (≥5 levels) met R² > 0.990; back‑calculated values were within ±40% at LOQ and ±20% for other levels per qualification criteria.
• Precision and accuracy: LOQ reproducibility assessed by 10 replicate injections yielded LOQ RSD <20% and LOQ bias <40% at LOQ level. Internal standard RSD acceptance was <30% across calibration curves.
• Robustness: Long sequence testing (40 matrix extracts, 70+ injections over 17 hours) with QCs injected every 10 samples showed stable quantitation and relative amount deviations within acceptance limits (typically <<40% at LOQ). No MS tuning or maintenance was required during testing.
• Orthogonal confirmation: The dual‑column approach delivered two consistent retention times (±0.1 min) plus accurate mass matching (±5 ppm) for precursor and optionally fragment ions obtained by in‑source fragmentation, enabling strengthened identification confidence without needing data‑dependent MS/MS.
• Practical limitations: Background contamination from ubiquitous laboratory PFAS (e.g., PFBA, PFPeA) constrained achievable LOQs in some lab environments; users must control blank and consumable contamination and set LOQs accordingly.

Benefits and practical applications

The workflow provides laboratories with:

• A turnkey HRMS solution that supports simultaneous targeted quantitation and suspect screening, simplifying future re‑interrogation when regulations or target lists evolve.
• Regulatory alignment with current EU drinking water criteria and the capacity to extend to proposed surface‑water standards and national compound lists.
• Compatibility with routine sample preparation techniques (SPE, DLLME) and throughput‑oriented UHPLC operation (14.5 min runtime).
• Robustness for routine monitoring across environmental matrices (drinking water, surface water, groundwater, sewage effluent).

Future trends and possibilities

Opportunities and likely developments include:

• Broader adoption of HRMS full‑scan workflows for routine PFAS monitoring, enabling integrated targeted and suspect screening with retrospective analysis as regulatory lists expand.
• Improved laboratory blank control and consumable qualification to push LOQs lower, particularly for ultra‑short chain PFAS that are prone to background contamination.
• Increased automation of sample concentration and cleanup (automated SPE/DLLME) coupled with HRMS data pipelines to increase throughput while maintaining confirmatory power.
• Integration of orthogonal confirmatory metrics (retention time on dual columns, accurate mass, isotope pattern, and in‑source fragments) into reporting frameworks to meet stricter regulatory confirmation requirements without routine MS/MS.

Conclusions

The described Orbitrap Exploris EFOX full‑scan workflow delivers a validated, robust approach for quantifying a comprehensive panel of 61 PFAS in methanolic extracts of water samples. It meets stringent linearity, precision and robustness criteria, provides orthogonal confirmation via dual‑column retention times and accurate mass, and supports retrospective/suspect screening. Practical sensitivity is sufficient when combined with common concentration methods (SPE, DLLME), but final LOQs should account for laboratory background PFAS. The workflow is a practical option for environmental and regulatory laboratories preparing for current and evolving PFAS monitoring requirements.

References

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3. Directive (EU) 2020/2184 of the European Parliament and of the Council on the quality of water intended for human consumption. 2020.
4. Johanson S, et al. Toxic tide rising: national approaches to address PFAS in drinking water across Europe. European Environmental Bureau. 2023.
5. US EPA. Drinking Water Research Methods.
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