Single Quadrupole LC-MS with Line-Shape Calibration Enhancement as an Economical Alternative for PFAS Screening

Significance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants of broad regulatory and public-health concern. Routine screening for a wide range of non-volatile PFAS typically relies on tandem mass spectrometry (TQ-LCMS). This study evaluates whether single quadrupole LC-MS (SQ-LCMS) enhanced by line-shape calibration and spectral-matching software can serve as an economical screening alternative that achieves low limits of quantitation (LOQs) and reliable compound confirmation for many regulated PFAS.

Objectives and overview of the study

The work aimed to develop and assess an SQ-LCMS workflow, augmented with MassWorks line-shape calibration and a Target Analysis function, to screen 40 environmentally relevant PFAS targets. Performance was compared to EPA expectations (Method 1633A) by: establishing calibration ranges, determining LOQs and instrumental detection limits (IDLs), evaluating mass and spectral accuracy under several calibration conditions, and examining practical suitability for routine screening.

Methodology

Standards and calibration

The investigators prepared 40 PFAS target analytes and 31 internal standards consistent with EPA Method 1633A concentration ranges. Neat standards in vials were analyzed across a wide calibration range (0.2–1560 ppb). LOQ criteria combined accuracy (70–130%), signal-to-noise >10, %RSD <20, and %RSE <20. IDLs were calculated from 11 replicates analogous to MDL procedures in CFR Appendix B to Part 136.

Data processing and calibration strategy

MassWorks (Cerno Bioscience) was applied to perform line-shape calibration and spectral accuracy evaluation. Multiple calibration/processing conditions were tested, varying the calibrants and acceptance criteria (e.g., inclusion of tune mix, benzenesulfonamide, sets of internal standards, target PFAS with S/N thresholds, and different mass and spectral accuracy cutoffs). The study compared how these conditions affected mass accuracy distribution and compound identifications.

Instrumentation used

Instrument platform and LC parameters

• LC-MS system: Shimadzu Nexera XR coupled to Shimadzu LCMS-2050 single quadrupole.
• Columns: Shim-pack Scepter C18-120, 3 µm; analytical 2.1 × 50 mm with a delay column 2.1 × 100 mm.
• Mobile phases: A = 2 mM ammonium acetate in water; B = acetonitrile.
• Flow rate: 0.4 mL/min; column oven 40 °C; injection volume 5 µL.

Ion source and MS settings

• Ionization: Electrospray ionization (ESI), negative mode implied by interface voltage -0.5 kV.
• Desolvation temperature 250 °C; DL (desolvation line) temperature 200 °C.
• Nebulizing gas 2.0 L/min; heating gas 7.0 L/min; probe position +2 mm.

Main results and discussion

Chromatography and detectability

A single chromatographic run resolved the 40 target PFAS across the gradient and produced adequate peak shapes at mid-range calibration concentrations. Grouped results for carboxylic acids, sulfonates/sulfonamides, and fluorinated telomer acids/sulfonates were reported showing LOQ and IDL values, and comparisons to EPA LOQs.

Limits of quantitation and detection

SQ-LCMS with line-shape calibration achieved LOQs comparable to EPA Method 1633A for many targets. IDLs derived from replicate analysis supported practical detection limits suitable for screening applications, provided criteria for accuracy, S/N and precision were met.

Mass accuracy and calibration condition effects

Mass accuracy distributions were strongly affected by the calibration/processing condition. Using a calibration that included a larger set of compounds (Condition 4: target PFAS with sufficient S/N) delivered the best overall mass accuracies. Stricter combined criteria (e.g., mass accuracy ≤10 mDa and spectral accuracy ≥95%) reduced false positives but also reduced the number of identified compounds, especially for low S/N species. Compounds with low S/N or mass accuracy worse than ~50 ppm were not reliably identified.

Role of spectral accuracy and internal standards

Applying spectral accuracy thresholds together with mass tolerance improved confidence in identifications from a unit-resolution instrument. Inclusion of multiple internal standards with known mass accuracy performance enhanced calibration robustness and contributed to better mass assignment across the target list.

Limitations identified

Despite improvements, SQ-LCMS remains less selective than TQ-LCMS or high-resolution MS: matrix interferences, coelution, and low-abundance analytes can limit reliable detection and confirmation. The approach depends on obtaining sufficient S/N and a representative set of calibrants and internal standards.

Practical benefits and applications

Benefits

• Lower capital and operational costs compared with TQ-LCMS or HRMS, enabling broader access for routine laboratories.
• Rapid screening capability for a broad PFAS panel when paired with line-shape calibration and spectral-matching software.
• LOQs and IDLs that are fit-for-purpose for many regulatory screening workflows when acceptance criteria are met.

Best-use cases

This workflow is well suited for laboratories performing large-volume screening, preliminary compliance checks, or prioritization of samples for confirmatory analysis by TQ-LCMS or HRMS. It can reduce the load on higher-end instruments by triaging samples that require confirmatory testing.

Future trends and potential uses

Emerging directions to increase applicability and robustness include:

• Improved software algorithms for line-shape calibration, spectral deconvolution and automated decision thresholds to increase identifications while controlling false positives.
• Expanded and more representative internal standard libraries (including isotopically labeled standards) to stabilize calibration across diverse matrices.
• Hybrid workflows that combine SQ-LCMS screening with targeted HRMS or TQ-LCMS confirmation in a tiered laboratory strategy.
• Integration of AI-driven pattern recognition and automated QC to detect matrix effects and adapt acceptance criteria dynamically.
• Extension of target lists and method validation across more complex matrices (biosolids, tissues) to align with regulatory frameworks.

Conclusion

This study demonstrates that single quadrupole LC-MS, when augmented with line-shape calibration (MassWorks) and a carefully designed calibration strategy, can provide an economical and effective screening method for many regulated PFAS. Using broader calibrant sets and combining mass accuracy with spectral accuracy criteria yielded the best identifications. The approach is not a full substitute for tandem or high-resolution confirmatory methods but is a practical screening tool for laboratories that require cost-effective, high-throughput PFAS surveillance.

References

1. U.S. Environmental Protection Agency. Method 1633A, Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS. January 2024.
2. Code of Federal Regulations. Appendix B to Part 136, Title 40 — Definition and Procedure for the Determination of the Method Detection Limit — Revision 2.