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Pharmacology & ToxicologybioRxiv

Novel PFAS in Alligator Blood Discovered with Non-Targeted Ion Mobility Spectrometry-Mass Spectrometry

Anna K. BoatmaniD, Gregory P. KudziniD, Kylie D. RockiD, Matthew P. Guillette, Frank Robb, Scott M. BelcheriD, Erin Shammel BakeriD

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a large and growing class of chemicals gaining global attention due to their persistence, mobility, and toxicity. Given the diverse chemical properties of PFAS and their varying distributions in water and tissue, monitoring of different matrices is critical to determine their presence and accumulation. Here, we used a platform combining liquid chromatography, ion mobility spectrometry, and high-resolution mass spectrometry for non-targeted analysis to detect and identify PFAS in alligator plasma from North Carolina (5 years, 2018-2022) and Florida (2021 only). Structures for 12 PFAS were elucidated, including 2 novel structures, and an additional 34 known PFAS were detected. Three of these compounds were previously unreported in environmental media. More PFAS were detected in North Carolina alligators and no novel PFAS were detected in Florida gators. Quantitative analysis of 21 of the known PFAS revealed that plasma concentrations did not change over the 5-year study. HighlightsO_LI46 PFAS were detected in blood plasma from North Carolina and Florida alligators using LC-IMS-HRMS C_LIO_LIStructures for 12 previously unreported PFAS were elucidated, including 2 novel structures C_LIO_LICape Fear River alligators had the most types and highest concentrations of PFAS C_LI

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In-depth summary

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (24 May 2025) · CC BY 4.0

Non-targeted LC-ion mobility-HRMS of North Carolina and Florida alligator plasma found 46 PFAS, including two new polyfluorinated triether acids; Cape Fear River animals carried the most.

Targeted mass spectrometry covers only a few dozen per- and polyfluoroalkyl substances (PFAS) with available standards, and non-targeted analysis (NTA) of biological matrices is hampered by abundant endogenous molecules, so bioaccumulating PFAS can be missed when only water is screened. Using long-lived apex-predator alligators downstream of a fluorochemical plant as sentinels, this work adds 5 years of plasma NTA data and reports PFAS absent from prior regional studies.

Key results

  1. ShownLibrary screening on collision cross section (CCS) and retention time (RT), combined with a non-targeted discovery workflow, identified 46 PFAS in alligator plasma. The discovery workflow alone produced candidate structures for 12 of them.

    Library v1 holds RT and CCS for 168 PFAS standards; screening matched 34 known PFAS (at least 45 molecules counting isomers), 21 of them quantified; discovery flagged 22 features: 12 candidate structures, 3 with fragment-only evidence, 5 unresolved; 46 PFAS in total. · Non-Targeted PFAS Analysis of Alligator Plasma; Target Screening and Absolute Quantification

  2. SuggestedTwo previously unreported H-substituted polyfluorinated triether acids (Compounds 592 and 628) were found in plasma and, retroactively, in archived Cape Fear River water data. They are proposed as the desulfonated and decarboxylated forms, respectively, of Compound 10, an ether previously found in water.

    m/z 592.9510 and 628.9180 with several overlapping LC peaks; shared MS2 fragments m/z 282.9822 and 262.9760 (HF loss) plus m/z 212.9792 (C4F7O2); co-eluting Compound 448 with matching drift time read as post-source decay of 592; parent Compound 10 not detected in alligators; Level 3. · H-substituted polyfluorinated triether acids; Conclusions

  3. SuggestedLong-chain ether sulfonates PFHxESA and PFHpESA, never previously detected in the Cape Fear River, were tentatively identified, along with a branched PFUdA isomer whose carboxylate sits mid-chain (co-br-PFUdA). The isomer was often as abundant as linear PFUdA or more.

    PFAESAs: RT and CCS of the most abundant peaks fit homologous-series trendlines from Donelson et al.; reported once before (European fish liver); shorter homologs and PFOESA absent; Level 3. co-br-PFUdA: RT 0.2 min earlier, M-H- CCS 0.9% smaller and M-H-CO2- CCS 0.9% larger than l-PFUdA (platform error <0.2%); Level 3. · Long Chain Perfluoroalkyl Ether Sulfonic Acids…; Novel Perfluoroundecanoic Acid Isomer…

  4. ShownThe quantified PFAS burden (sum of 21 PFAS) was highest and most dominated by sulfonic acids in Cape Fear River (CFR) alligators, compared with Lake Waccamaw (LW) and Florida (FL) animals. Neither CFR nor LW showed a significant change from 2018 to 2022.

    Σ21 average 249.1 ng/ml in CFR (median 225.2; range 17.3 to 806.5) vs 42.1 ng/ml in LW (median 33.1) and 31.8 ng/ml in FL (median 30.7); CFR median 84.9 % PFSAs; site association p < 0.0001; no trend in CFR (F 0.7962, DF 96, p 0.374) or LW (F 1.301, DF 72, p 0.258). · Spatial and Temporal Trends; Statistical Analysis

  5. SuggestedOther PFAS that lack standards or are rarely monitored were also detected: a new Nafion Byproduct 2 isomer, fipronil sulfone, unsaturated PFOS isomers, PFPeDA and PFUdS. PFPeDA and PFUdS had not been reported in the Cape Fear River before.

    NB2 A and B at Levels 2 and 1; PolyFESA D (NB2 isomer new to environmental samples) Level 3; Unknown PolyFESA C Level 5; fipronil sulfone, PFPeDA (15-carbon PFCA) and PFUdS (11-carbon PFSA) Level 2; U-PFOS isomers Level 3; fragments hinted at further perfluoroether carboxylic acids lacking precursor signal. · Other PFAS Identified Using NTA; Non-Targeted PFAS Analysis of Alligator Plasma

Methods, data and reproducibility

Acetonitrile-precipitated plasma (50 µl) was analyzed by reversed-phase LC, drift-tube ion mobility and QTOF HRMS (negative ESI). It was screened against a CCS and retention-time PFAS library, then mined with Fluoromatch-IM homologous-series/mass-defect scoring and size-dependent all-ions fragmentation.

  • Alligator plasma, lower Cape Fear River sites (Greenfield Lake, Orton Pond, Oak Island, Bald Head Island), 2018 to 2022 · alligator · n = 98 · MassIVE MSV000097330 (raw LC-IMS-HRMS files, all sites)
  • Alligator plasma, Lake Waccamaw reference site in the Lumber River basin, North Carolina · alligator · n = 74
  • Alligator plasma, St. John's River, Florida (no known fluorochemical manufacturing), 2021 only · alligator · n = 26
  • NIST SRM 1957 human serum used as extraction quality control · human · n = three QCs per extraction batch
  • Matrix-matched calibration curves for 26 native PFAS in charcoal-stripped fetal bovine serum (0.128 to 4,000 ng/ml) · bovine (fetal bovine serum) · n = seven concentrations
  • Baker lab LC-IMS-MS PFAS library v1 with RT and CCS values acquired under identical conditions · n = 168 PFAS standards
  • Archived Cape Fear River passive-sampler water NTA data files from Donelson et al., screened retroactively
Methods & tools
LC-IMS-CID-HRMS (Agilent drift-tube IMS with QTOF, JetStream ESI negative mode), Size-dependent all-ions fragmentation (AIF) method (Donelson et al.), PNNL Preprocessor v4.0, Agilent IM-MS Browser v10.0, Skyline-daily v24.1.1, Agilent Mass Profiler v10.0.2.202, Fluoromatch-IM v1.2, Baker lab LC-IMS-MS PFAS library v1, In-house Python feature-filtering software, GraphPad Prism v10.3.1, PubChem formula search, Spearman correlation and multiple linear regression
Data & accessions
https://panoramaweb.org/2025-alligator-PFAS.url
Reagents & environment
PNNL Preprocessor v4.0; Agilent IM-MS Browser v10.0; Skyline-daily v24.1.1; Agilent Mass Profiler v10.0.2.202; Fluoromatch-IM v1.2; GraphPad Prism v10.3.1; Python 3.12; Agilent Zorbax Eclipse Plus C18 column
Reproducibility gaps
No code release is stated for the in-house Python feature-filtering software (±10 ppm, 2.0% CCS cutoffs). Full extraction, instrument and Fluoromatch settings are left to the Supplementary Materials.
Limitations (6)
  • Detection limits were poor for several PFAS classes, such as perfluoroether carboxylic acids, and the absence of internal fragments for some candidate structures prevented full identification of several flagged molecules. · stated by the authors
  • Only 21 PFAS were quantified. The other library hits (standards exist for 13 of them) and every discovery-workflow compound were left unquantified, so the Σ21 sums understate total PFAS burden. · stated by the authors
  • Adults and juveniles were pooled, despite a significant interaction between life stage and body mass index (p < 0.0001), to keep n ≥ 3 per site and year. Florida was sampled in a single year, so no time trend could be tested there. · stated by the authors
  • Prioritizing NTA features can inherently drop some molecules. Data analysis is slow and resource-intensive, and extracts cannot always be re-run once a feature of interest turns up. · stated by the authors
  • The switch to plasma from the serum used in the 2018-2019 study relies on unpublished pilot work showing no detection difference. Whether results are comparable with the earlier targeted alligator data is therefore not directly demonstrated. · noted in this summary
  • Cape Fear River sub-sites differed by year (Orton Pond and Oak Island sampled only in 2019, Bald Head Island in 2019 and 2022). Pooled temporal regressions may therefore mix site effects with year effects. · noted in this summary

Who should read:Read this if you do non-targeted PFAS or exposomics work with ion mobility mass spectrometry, or you monitor wildlife or human PFAS exposure near the Cape Fear River fluorochemical discharge.

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