Feasibility for a PFAS Screening Protocol by Matrix- Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants of growing regulatory and analytical concern. Routine LC-MS/MS workflows used for trace PFAS quantification are highly sensitive but also vulnerable to carryover and contamination by high-concentration samples, which can cause instrument downtime and false results. A rapid pre-screen that flags samples with elevated PFAS levels prior to LC-MS/MS can reduce contamination risk, save time and costs, and streamline laboratory throughput. The evaluated MALDI-TOF approach aims to provide a fast, low-preparation screening step compatible with existing sample preparation paths (notably after SPE reconstitution in methanol).

Objectives and study overview

The study assessed feasibility of using a benchtop linear MALDI-TOF mass spectrometer (Shimadzu MALDI-8030) as a semi-quantitative screening tool for PFAS in aqueous samples. Key goals were to determine whether MALDI-TOF signal intensity/area can indicate samples that exceed LC-MS/MS upper calibration limits (requiring dilution), to evaluate lower limits of quantification and linearity for representative PFAS (PFOS and PFOA), and to test practical sample/matrix combinations that provide reproducible spot homogeneity on FlexiFocus slides.

Methodology

Samples: PFOS and PFOA standards were prepared from concentrated stocks and diluted into either water or 100% methanol. An isotopically labelled internal standard (13C-PFOS) was added to give 10 ng/mL final in analysed aliquots. Spotting: Aliquots were mixed 1:1 with MALDI matrices and spotted in triplicate (0.5 and 1 µL) onto 96-well FlexiFocus slides to improve spot homogeneity; about 180 spots were analysed.

Matrices and solvents: Two matrices were evaluated — norharmane (NRM) and 1,8-bis(tetramethylguanidino)-naphthalene (TMGN) — prepared at various concentrations in MeOH (NRM) or 70:30 ACN:H2O (TMGN) for aqueous tests, and in 100% MeOH for methanolic preparations. Acquisition: Negative polarity linear MALDI-TOF acquisition across m/z 250–1000 using 5 shots/profile at 200 Hz and pulsed extraction optimized for the MALDI-8030.

Calibration and evaluation: Calibration curves were constructed across multiple concentration ranges tailored to each experiment: low range (1 ppt–10 ppb), mid-range (500 ppt–100 ppb) and high-range relevant to LC-MS/MS upper limits (e.g., 62.5 ppb per EPA1633). Linearity was evaluated by R2 values from intensity/area versus concentration; limits of quantification and loss of linearity at low concentrations were assessed. Representative spectra were inspected to confirm peak identity and signal behavior.

Used instrumentation

• MALDI-8030 benchtop linear MALDI-TOF mass spectrometer (Shimadzu).
• FlexiFocus 96-well slides (Shimadzu) for improved spot homogeneity.
• Matrices: norharmane (NRM) and 1,8-bis(tetramethylguanidino)-naphthalene (TMGN).
• Isotopically labelled internal standard: 13C-PFOS.

Main results and discussion

Matrix performance: TMGN outperformed NRM in linearity for PFOS when samples and matrix were prepared in methanol. Optimal matrix concentrations were 0.5 mg/mL for TMGN and 0.1 mg/mL for NRM on FlexiFocus slides.

PFOS screening performance: When samples and matrix were prepared in 100% methanol and spotted on FlexiFocus slides, PFOS showed linear response from approximately 500 ppt up to at least 100 ppb (R2 ≈ 0.94 for 500 ppt–100 ppb), which encompasses the LC-MS/MS upper limit relevant for EPA1633 (62.5 ppb). Below ~500 ppt (in aqueous preparations) the response flattened and lost linearity, likely due to sample-phase effects (surface partitioning of PFAS in aqueous droplets) and variable sampling from bulk water. Use of methanol and FlexiFocus slides improved precision and spot behavior, but did not restore linearity below the observed low-concentration threshold in practice.

PFOA screening performance: In methanolic preparations with 0.5 mg/mL TMGN, PFOA showed good linearity only above ~5 ppb; below 5 ppb linearity was lost. Across 5–100 ppb, R2 approached 0.99, allowing confident identification of samples exceeding LC-MS/MS upper quantitation limits.

Implications: The MALDI-TOF approach can serve as a rapid semi-quantitative screen for post-SPE reconstituted samples in methanol to detect those exceeding LC-MS/MS ULOQs and thus requiring dilution. However, direct screening of raw aqueous samples is unreliable at low ppt concentrations due to sampling variability and matrix effects. Typical lower practical screening thresholds observed were ~500 ppt for PFOS and ~5 ppb for PFOA.

Benefits and practical applications of the method

• Rapid pre-screening: Minimal sample prep and fast acquisition allow high-throughput checking of samples prior to LC-MS/MS, reducing risk of instrument contamination and carryover from high-concentration PFAS samples.
• Resource savings: Identifying samples that require dilution ahead of LC-MS/MS reduces wasted instrument time, reruns and potential maintenance costs.
• Compatibility with SPE workflows: Best applied after SPE and reconstitution in methanol, aligning with common PFAS laboratory protocols.
• Semi-quantitative triage: Sufficient for flagging samples above LC-MS/MS ULOQ (e.g., ~62.5 ppb) though not suitable as a full replacement for trace quantitation by LC-MS/MS.

Future trends and potential uses

• Matrix and surface engineering: Further optimization of matrices, solvents and slide chemistries could push lower limits of linearity and reproducibility, possibly enabling more sensitive aqueous screening.
• Automation and integration: Automated spotting, spectral processing and decision rules could integrate MALDI-TOF screening into routine PFAS lab pipelines to gate LC-MS/MS runs automatically.
• Expanded analyte panels: Evaluation of additional PFAS homologues and mixed-class screening could broaden applicability to routine environmental monitoring.
• Hybrid workflows: Combining rapid MALDI-TOF triage with targeted LC-MS/MS confirmation provides an efficient two-tiered approach for high-throughput laboratories.

Conclusion

Linear MALDI-TOF on an entry-level bench-top instrument (MALDI-8030) can be used as a fast, low-preparation screening tool for PFAS in samples prepared in methanol after SPE. The method reliably flags samples with PFOS in the range ~500 ppt–100 ppb and PFOA above ~5 ppb, including identification of samples exceeding LC-MS/MS ULOQs relevant to standard protocols (e.g., EPA1633). Limitations include loss of linearity at low concentrations and unreliable results for direct aqueous sampling; therefore, the recommended use is post-SPE methanolic reconstitution as a triage step to protect LC-MS/MS workflows.

References

1. Jones CJ. Detection of PFAS in Aqueous Samples by Matrix Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry. Shimadzu Application News No. 12-MO-543-EN.
2. Tighe M, et al. Rapid PFAS removal from water with floating polymer assisted by air bubbles. Chemosphere. 2025.