Tackling PFAS Complexity with HRMS and Bioanalytical Techniques

Per- and polyfluoroalkyl substances (PFASs) are highly persistent anthropogenic compounds that are widespread in the environment. There are thousands of PFASs, yet few neat standards exist for unequivocal identification, quantification, or toxicity assays. The bioanalytical study of complex commercial PFAS mixtures is an innovative route to better understand novel PFAS exposure and toxicity. Here, we highlight efforts using high-resolution mass spectrometry (HRMS) and exposure-relevant mixtures to prioritize PFASs based on their potential to accumulate in living organisms.

Per- and polyfluoroalkyl substances (PFASs) are unlike many legacy hydrophobic persistent organic pollutants (POPs) in that fatty tissues are not their main reservoir in the body. Many PFASs are anions at physiological pH, and they have similar structures to endogenous fatty acids. Fatty acids are transported throughout the body by the transporter protein serum albumin, and so are PFASs. Although the bioaccumulation of legacy POPs can be predicted by hydrophobicity alone, PFAS toxicokinetics in the human body are related to affinity for human serum albumin (HSA) and renal transporters (organic anion transporters, or OATs), as well as affinity for the phospholipids that make up cellular membranes. These complex interactions have necessitated the development of more sophisticated models to predict and understand PFAS toxicokinetics (1), as well as multiple in vitro and in vivo experimental approaches to ground truth them (2).

Analytical Challenges to Comprehending PFAS Body Burden

The observation that “trace amounts of organic fluorocompounds derived from commercial products” were present in blood from the general population was first made in the 1970s using nuclear magnetic resonance (NMR) (3). Since then, our ability to identify and measure PFASs in biological samples has advanced significantly. Presently, liquid chromatography with tandem mass spectrometry (LC–MS/MS) is recognized as the go-to method for sensitive and accurate measurement of targeted PFASs, with detection limits of 0.1–1 ng/mL, depending on the analyte. Using these methods has revealed that PFASs are present in the blood of virtually every human on Earth, as well as most wildlife. Although the levels of PFASs are often low (near detection limits), they have been found to be elevated in countless communities with PFAS-impacted drinking water and in occupationally exposed individuals. The health consequences of these exposures are concerning, including immunosuppression, several types of cancer, increased risk of obesity, and liver disease.

LC–MS/MS has revealed that perfluoroalkyl acids (PFAAs), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are widespread in human blood. The coupling of this technique with integrative organofluorine measurement techniques like total organofluorine combustion ion chromatography (TOF-CIC) has also highlighted how little of the total organofluorine body burden is accounted for by these targeted LC–MS/MS methods. A recent study monitoring the blood of the general Swedish population found that an average of 60% and 41% of total organofluorine was unexplained by targeted LC–MS/MS in females and males, respectively (4). The data raised the following question: what compounds make up the rest of this organofluorine? Are they fluorinated pharmaceuticals, novel PFASs with no analytical standards, or ultra-short-chain or neutral volatile PFASs not caught by traditional reverse-phase LC (RPLC)?

There are thousands of PFASs with diverse properties that pose a considerable challenge for analytical chemists attempting to characterize all PFASs contributing the unidentified organofluorine burden in a biological sample. In addition, some PFASs (referred to as “precursors” or “pre-PFAAs”) are labile under certain conditions, transforming in the environment or in vivo to form perfluorinated end products that are highly stable. Many PFAS precursors eventually form common perfluoroalkyl acids, where original precursor functionality and structure have been lost. This complicates the understanding of source contribution or environmental forensics because it is impossible to know which precursor was initially present. This also precludes an understanding of the full health impacts because direct exposure to a distinct precursor is expected to have different toxicological effects than exposure to the final product PFAA (5).

We know that humans are chronically exposed to a complex mixture of PFASs, including poorly understood pre-PFAAs that are widespread in commercial products. Because of studies combining LC–MS/MS and integrative techniques like TOF-CIC, we also know that we are not capturing total organofluorine with targeted LC–MS/MS techniques. Without confirming molecular identification, it is impossible to fully understand the predominant exposure sources and the associated health risks.

Bioanalytical Strategies for Prioritizing Novel PFASs

The use of complex, exposure-relevant mixtures enables the study of novel PFASs present in these mixtures when there are no neat standards. This allows for the prioritization of novel PFASs based on their bioaccumulation or biotransformation to known toxic PFAAs. Yeung and Mabury demonstrated the utility of such methods in 2013 by conducting detailed analyses of rainbow trout tissues after exposure to two distinct aqueous film-forming foams (AFFFs) (6). AFFFs are used to fight fuel fires, and they were widely used as part of fire training activities by first responders and military personnel. Prolonged use of AFFFs during training or simulation exercises have left large impacted fire training areas where the soils, groundwater (7), and surrounding coastal environments (8) will remain contaminated for decades to centuries to come (9). Yeung and Mabury noted that not only did targeted PFASs measurable by LC–MS/MS accumulate in the AFFF-exposed fish, but the fish also contained significant amounts of unknown organofluorine.

Several recent studies have demonstrated the usefulness of similar techniques incorporating high-resolution mass spectrometry (HRMS) and in vitro or in vivo exposures to complex AFFF mixtures for investigating the bioaccumulation and toxicity of PFAS-containing AFFFs. Yang and associates (10) used size-exclusion column co-elution and HRMS to measure the binding potential of PFASs to human liver fatty acid binding protein (hL-FABP) and identify novel PFASs in a commercial AFFF mixture and in AFFF-impacted waters that are hL-FABP ligands. They identified novel substituted perfluoroalkyl sulfonates (PFSAs) including unsaturated, ketone-, hydrogen-, chlorine-, and oxygen-substituted PFSAs, as hL-FABP ligands. Li and colleagues (11) combined HRMS and equilibrium dialysis to identify AFFF-associated PFASs with high bioaccumulation potential. They tentatively identified several of the novel substituted PFSAs also found by Yang and associates that were likely noncovalently bound to HSA, as well as at least one novel AFFF-associated sulfonamide precursor (N-dimethylammoniopropyl perfluorohexane sulfonamide) that appeared to have some potential for covalently binding to HSA.

Recent in vivo treatments using AFFF have also been used for determining bioaccumulation potential and biotransformation products of novel PFASs. Our work applying HRMS to analyze serum from mice dosed with a field-collected predominantly electrochemically fluorinated AFFF (the same AFFF used by Li and colleagues in HSA binding studies) also identified several novel substituted PFSAs accumulating in blood serum after a brief 6 d depuration, confirming that these compounds highlighted by in vitro studies were indeed accumulating in AFFF-exposed living organisms (12). We also identified a series of novel bis-sulfonamides in post-depuration mouse serum, though these compounds were neither detected in the original AFFF used in our dosing study, nor have they been recognized as a potential component of AFFF in other HRMS studies. Potential explanations for the presence of these bis- sulfonamides in AFFF-dosed mouse serum where these compounds may have been present in the AFFF at a low level and were not detectable until their accumulation in mouse serum enhanced their relative abundance in comparison with other AFFF components. This highlights their bioaccumulation potential, or they formed as metabolic products of other sulfonamide-based AFFF components. The discovery of these compounds in an in vivo AFFF-dosed model highlights the usefulness of such methods for prioritizing identification of novel biologically relevant PFASs that may be hidden in complex mixtures prior to exposure.

Our later work using HRMS to characterize PFAS mixtures in human blood serum from an AFFF-impacted community in El Paso County, Colorado, further highlights the utility of these laboratory-based complex mixture studies to inform and direct human biomonitoring. Among the approximately 200 residents whose blood was analyzed in this study, unsaturated PFOS (UPFOS), a compound tentatively identified with high bioaccumulation potential in our mouse experiments, was detected in 85% of residents, and it was also found in raw drinking water (13). The spatial trends in semi-quantitative abundance suggested this compound originated from the same AFFF source as other PFASs in El Paso County drinking water, and the widespread presence of UPFOS in human serum echoed our findings of high bioaccumulation potential in AFFF-exposed mice.

Similar methods have recently been applied to prioritize novel compounds in complex PFAS-containing environmental samples. Bangma and associates (14) treated mice with industrially impacted surface water from Bladen County, North Carolina, a site previously impacted by Chemours’ Fayetteville Works where elevated detections of several novel ether PFASs were found. Their results corroborated recently characterized human serum profiles from the same area (15), highlighting PFO5DA, Nafion Byproduct 2, and HydroEVE as compound needing prioritization for toxicological testing.

Future Outlook for PFAS Analysis in Biological Matrices

Despite the application of many advanced analytical techniques and the work of numerous investigators worldwide, the significant portion of unidentified organofluorine remaining in the biosphere presents a significant challenge in the analytical community. Although techniques combining HRMS with in vitro or in vivo prioritization of complex, exposure-relevant dosing mixtures hold promise, there are still many uncertainties inherent in these approaches. For example, these studies may be influenced by saturation of binding sites, competitive binding amongst individual PFASs, or both. Such saturation and competition are likely not representative of real exposure scenarios occurring at lower concentrations, but elevated dosages are needed to facilitate identification of novel compounds. Additionally, measuring PFASs requires an expensive, time-intensive methodology such as LC–HRMS, and the uncovering of novel PFASs will likely require additional method development and even more advanced analytical instrumentation to capture sub-classes like the ultra-short-chain and neutral volatile PFASs. Despite these many challenges, continual progress is being made to understand total PFAS body burden, including advances in sample preparation, instrumentation, and in silico techniques to broaden the fraction of total PFASs that are encompassed. Additionally, efforts to harmonize naming conventions, provide machine-readable structures for novel PFASs (16), and communicate confidence of novel PFAS identifications in a consistent way (17), as well as the growing number of PFASs identified and entered into spectral databases, are accelerating discovery and understanding of the biological relevance of novel PFASs.

References

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Carrie McDonough is an Assistant Professor in the Department of Chemistry at Carnegie Mellon University in Pittsburgh, Pennsylvania. Wesley Scott is a Postdoctoral Research Associate in the Department of Chemistry at Carnegie Mellon University in Pittsburgh, Pennsylvania. Direct correspondence to: wesleys@andrew.cmu.edu