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 sampled alligators from the Cape Fear River in North Carolina over 5 years (2018-2022) to search for novel PFAS and monitor PFAS levels in blood. Using a platform combining liquid chromatography, ion mobility spectrometry, and high-resolution mass spectrometry (LC-IMS-HRMS), we applied a non-targeted analysis (NTA) method to detect and identify PFAS in the alligators. Structures for 12 PFAS were elucidated, including 2 novel structures, and an additional 34 known PFAS were detected. 3 of these compounds were previously unreported in environmental media. Quantitative analysis of 21 of the known PFAS revealed that plasma concentrations did not change in alligator plasma over the 5 year study, indicating that exposure is ongoing for these animals.

Our NTA platform combining liquid chromatography, ion mobility spectrometry, collision induced dissociation, and high-resolution mass spectrometry (LC-IMS-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. In this study, we first searched for PFAS in each alligator plasma sample by collecting precursor-only (or MS1-only) full scan data to maximize feature abundance. Next, we collected fragmentation (or MS2) data using size-dependent all-ions fragmentation (AIF) to elucidate unknown structures. The data was then analyzed using complementary approaches combining a library screening workflow to find known PFAS and a discovery workflow to uncover additional PFAS not present in the library.

Samples were alligator plasma extracted in acetonitrile. Plasma samples were collected from alligators in three watersheds over the 5 year study (2018-2022): the Cape Fear River downstream of a fluorochemical manufacturer in North Carolina (CFR, n = 98); Lake Waccamaw, 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, 2021 only). Within the CFR, four distinct locations were sampled: Greenfield Lake (2018-2022), Orton Pond (2019 only), Oak Island (2019 only), and Bald Head Island (2019 and 2022).