Identifying PFAS in Alligator Plasma with LC–IMS-HRMS
A combination of liquid chromatography ion mobility spectrometry, and high-resolution mass spectrometry (LC–IMS-HRMS) for non-targeted analysis (NTA) was used to detect and identify per- and polyfluoroalkyl substances (PFAS) in alligator plasma.
Due to the diverse chemical properties of per- and polyfluoroalkyl substances (PFAS), as well as their fluctuating distributions in water and tissue, the monitoring of different matrices is critical to determine their existence and accumulation. A research team made up of associates of the University of North Carolina (Chapel Hill, North Carolina), Clemson University (Clemson, South Carolina), North Carolina State University (Raleigh, North Carolina), and Environmental Education, Awareness, Research, Support & Services (Titusville, Florida) set out to identify as many PFAS as possible in alligator plasma with a focus on the 83 novel and uncommon compounds that could go undetected in non-targeted analysis (NTA) of water. A combination of liquid chromatography ion mobility spectrometry, and high-resolution mass spectrometry (LC–IMS-HRMS) was used in their study. A paper based on their five-year study was published on the preprint repository bioRxiv (1).
Much research has been conducted concerning the presence of PFAS in drinking and surface water using both targeted and non-targeted analyses (2–6), the latter used when standards are not available or the study of new PFAS is desired (1). NTA efforts in water and biological tissue have shown that different PFAS are detected in different matrices (7–9), which increases the importance of testing both the exposure source and the exposed organism (7,10). NTA of complex biological matrices, however, can be challenging, as obtaining biological samples often requires time- and resource-intensive collection processes. Furthermore, the samples to be analyzed may have many other molecules present, such as proteins, lipids, and metabolites, resulting in feature lists dominated by highly abundant biological molecules that have no relevance to the analytes of interest. Work to improve and increase NTA analyses in biological samples, therefore, is of the utmost importance for contaminant studies, including PFAS (1).
The lower portion of North Carolina’s Cape Fear River (CFR) acts as a primary source of drinking water for the local populace and animal residents (1). Famously located on the river is a fluorochemical manufacturer of fluorochemicals that produces the sulfonated tetrafluoroethylene-based fluoropolymer-copolymer Nafion (11) using perfluoroether compounds; previous non-targeted studies in this region showed perfluoroether presence in seabirds (7), in addition to other unknown fluorochemicals in water (6). Targeted, quantitative studies in the CFR have noted high PFAS levels in water (2,6,12), fish and other native wildlife (7,13), human serum (5,14), and domestic species such as horses and dogs (15).
Due to the alligator’s long life span, their position at the top of the environment’s food chain (both of which result in high levels of exposure to aquatic bioaccumulative pollutants), and their complex and robust immune systems (which make them sensitive predictors for exposure to immunotoxic chemical pollutants such as PFAS), the researchers decided that the presence of these compounds in these reptiles would be cause for concern regarding human exposure (1). Plasma samples were collected from alligators in three watersheds throughout the study: the aforementioned Cape Fear River downstream of a fluorochemical manufacturer in North Carolina (CFR, n = 98); Lake Waccamaw, North Carolina, a nearby reference site in the Lumber River basin with no known PFAS point sources (LW, n = 74); and a region in Florida with no known fluorochemical manufacturing (FL, n = 26). Note that the North Carolina specimens were collected over the five years that the study was conducted, whereas the Florida samples were collected in 2021 only. A NTA testing regimen combining LC–IMS-collision induced dissociation (CID)-HRMS was used to evaluate the extracted plasma samples; the multiple separations possible with this NTA platform enable the evaluation of known and new PFAS with high identification confidence (1).
Structures for 12 PFAS were elucidated from the samples, which included two novel structures; an additional 34 known PFAS were detected (three of which were previously unreported in environmental media). More PFAS were detected in the samples collected in North Carolina than those collected in Florida, and no novel PFAS were detected in the Florida subjects. Quantitative analysis of 21 of the known PFAS revealed that plasma concentrations did not change throughout the five-year study, indicating that exposure is ongoing (1).
The authors note that they chose to group adults and juveniles together in their research to ensure sufficient samples for each site and year were available, and admit that a larger sample size of both age groups would be beneficial for future work; however, catching even one alligator can be a challenge, and, in their own words, “sometimes you have to make the best of what you get” (1).
Alligator in a pond. © Pam - stock.adobe.com
References
1. Boatman, A. K.; Kudzin, G. P.; Rock, K. D.; et al. Novel PFAS in Alligator Blood Discovered with Non-Targeted Ion Mobility-Mass Spectrometry. bioRxiv 2025, 644452. DOI: 10.1101/2025.03.20.644452
2. Sun, M.; Arevalo, E.; Strynar, M.; et al. Legacy and Emerging Perfluoroalkyl Substances are Important Drinking Water Contaminants in the Cape Fear River Watershed of North Carolina. Environ. Sci. Technol. Lett. 2016, 3 (12), 415–419. DOI: 10.1021/acs.estlett.6b00398
3. McCord, J.; Strynar, M. Identification of Per- and Polyfluoroalkyl Substances in the Cape Fear River by High Resolution Mass Spectrometry and Nontargeted Screening. Environ. Sci. Technol. 2019, 53 (9), 4717–4727. DOI: 10.1021/acs.est.8b06017
4. Kirkwood-Donelson, K. I.; Dodds, J. N.; Schnetzer, A.; et al. Uncovering Per and Polyfluoroalkyl Substances (PFAS) with Nontargeted Ion Mobility Spectrometry-Mass Spectrometry Analyses. Sci. Adv. 2023, 9 (43), eadj7048. DOI: 10.1126/sciadv.adj7048
5. Kotlarz, N.; Guillette, T.; Critchley, C.; et al. Per- and Polyfluoroalkyl Ether Acids in Well Water and Blood Serum from Private Well Users Residing by a Fluorochemical Facility near Fayetteville, North Carolina. J. Expo. Sci. Environ. Epidemiol. 2024, 34 (1), 97–107. DOI: 10.1038/s41370-023-00626-x
6. Weed, R. A.; Campbell, G.; Brown, L.; et al. Non-Targeted PFAS Suspect Screening and Quantification of Drinking Water Samples Collected through Community Engaged Research in North Carolina's Cape Fear River Basin. Toxics 2024, 12 (6), 403. DOI: 10.3390/toxics12060403
7. Robuck, A. R.; McCord, J. P.; Strynar, M. J.; et al. Tissue-Specific Distribution of Legacy and Novel Per- and Polyfluoroalkyl Substances in Juvenile Seabirds. Environ. Sci. Technol. Lett. 2021, 8 (6), 457–462. DOI: 10.1021/acs.estlett.1c00222
8. Pickard, H. M.; Ruyle, B. J.; Thackray, C. P.; et al. PFAS and Precursor Bioaccumulation in Freshwater Recreational Fish: Implications for Fish Advisories. Environ. Sci. Technol. 2022, 56 (22), 15573–15583. DOI: 10.1021/acs.est.2c03734
9. De Silva, A. O.; Armitage, J. M.; Bruton, T. A.; et al. PFAS Exposure Pathways for Humans and Wildlife: A Synthesis of Current Knowledge and Key Gaps in Understanding. Environ. Toxicol. Chem. 2021, 40 (3), 631–657. DOI: 10.1002/etc.4935
10. Bangma, J.; Pu, S.; Robuck, A.; et al. Combined Screening and Retroactive Data Mining for Emerging Perfluoroethers in Wildlife and Pets in the Cape Fear Region of North Carolina. Chemosphere 2024, 363, 142898. DOI: 10.1016/j.chemosphere.2024.142898
11. Nafion; Wikipedia.https://en.wikipedia.org/wiki/Nafion (accessed 2025-04-08).
12. PFAS Testing Results; Cape Fear Public Utility Authority. https://www.cfpua.org/833/PFAS-Testing-Results (accessed 2024-12-20).
13. Guillette, T. C.; McCord, J.; Guillette, M.; et al. Elevated Levels of Per- and Polyfluoroalkyl Substances in Cape Fear River Striped Bass (Morone saxatilis) are Associated with Biomarkers of Altered Immune and Liver Function. Environ. Int. 2020, 136, 105358. DOI: 10.1016/j.envint.2019.105358
14. Kotlarz, N.; McCord, J.; Collier, D.; et al. Measurement of Novel, Drinking Water-Associated PFAS in Blood from Adults and Children in Wilmington, North Carolina. Environ. Health Perspect. 2020, 128 (7), 77005. DOI: 10.1289/EHP6837
15. Rock, K. D.; Polera, M. E.; Guillette, T. C.; et al. Domestic Dogs and Horses as Sentinels of Per- and Polyfluoroalkyl Substance Exposure and Associated Health Biomarkers in Gray’s Creek North Carolina. Environ. Sci. Technol. 2023, 57 (26), 9567–9579. DOI: 10.1021/acs.est.3c01146
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