PFOS is one of the most scrutinized members of the PFAS family – but in most environmental testing, it is still reported as a single total value. That simplicity may be hiding important chemistry. In a recent paper, Diana Aga and colleagues at the University at Buffalo used UHPLC-cIMS-qTOF-MS to separate six PFOS isomers. While branched PFOS made up more than half of the total signal in wastewater samples, linear PFOS dominated avian egg yolks, accounting for more than 88 percent of the total. The study also found major differences in ionization efficiency between isomers, with some branched PFOS species producing signals two to five times stronger than the linear form.
Here, Aga, SUNY Distinguished Professor and Henry M. Woodburn Chair in the Department of Chemistry, and Jenise Paddayuman, a PhD Student, both at the University at Buffalo, USA, explain what drew the team to PFAS isomers, why cyclic ion mobility proved so useful, and what isomer-specific data could mean for future PFAS risk assessment.
What inspired this work? What first made you start thinking about the importance of PFAS isomers?
We are aware that many PFAS are produced as mixtures of branched and linear isomers, which are observed in environmental samples, as in the case of PFOS. Even with conventional LC-MS/MS for PFAS analysis, we can detect overlapping peaks from branched PFOS that elute before linear PFOS. However, we observed that the relative intensities of these branched isomers vary significantly between different matrices such as wastewater, fish, and human blood, suggesting that branched isomers may have different environmental fate and environmental stability than the linear isomers. Interestingly, most studies report only the total concentration of each PFAS; for instance, only the total PFOS or total PFOA concentrations are required for regulatory reporting to comply with the maximum contaminant levels established by the US EPA.
On the contrary, regulatory agencies require the identification and quantification of toxicologically relevant isomers, metabolites, and impurities when establishing residue definitions and maximum residue levels for pesticides and pharmaceuticals. For instance, the U.S. Food and Drug Administration (FDA) requires the evaluation of individual drug isomers because stereoisomers can differ markedly in potency, receptor binding, metabolism, and toxicity. These regulatory frameworks show that chemically similar substances can lead to significantly different biological outcomes. However, this understanding has not yet been fully applied to contaminants of emerging concern, such as PFAS in the environment and among humans. Currently, monitoring and regulation often rely on aggregated metrics of total PFAS, creating a critical disconnect between the science of isomer resolution and decision-making processes for water quality and human health.
Which analytical techniques were most important for distinguishing between linear and branched PFAS isomers in real samples, and why?
Cyclic ion mobility spectrometry (cIMS) was the most critical technique for distinguishing linear and branched PFAS isomers in real samples, as it provides a powerful, orthogonal separation dimension to conventional UHPLC-qTOF-MS. When coupled as UHPLC-cIMS-qTOF-MS, it enables resolution of isomers that often co-elute chromatographically and are indistinguishable by mass spectrometry alone. By allowing ions to traverse the mobility cell multiple times, cIMS effectively extends the separation path length and significantly enhances resolving power. This allows separation of compounds with subtle differences in molecular size and three-dimensional shape. Although PFAS isomers share identical molecular formulas, branched isomers are generally more compact, while linear isomers adopt more extended conformations. These structural differences translate into distinct drift times and collision cross-section (CCS) values, providing a reliable basis for isomer discrimination. While other approaches, such as specialized LC columns, extended-column GC-MS, and supercritical fluid chromatography (SFC), can improve isomer separation, cIMS stands out because it directly probes gas-phase ion structure, offering enhanced selectivity and confidence in identifying PFAS isomers in complex environmental matrices.
As you followed PFOS from water to fish and then to bird eggs, what patterns stood out most to you – and were any of them unexpected?
One of the most notable observations was the systematic shift in PFOS isomer distributions across the environmental continuum from water to fish and ultimately to avian egg yolks. Specifically, we observed differential enrichment of linear versus branched isomers at each trophic level, suggesting isomer-specific partitioning, bioaccumulation, and maternal transfer processes. While these trends were not entirely unexpected, they provide strong empirical support for our hypothesis that PFAS isomers exhibit distinct environmental fate and transport behaviors. This finding is particularly compelling because it highlights the importance of resolving isomeric composition when assessing bioaccumulation and trophic transfer of PFOS in aquatic food webs.
What do your results suggest about the processes that control how different PFOS isomers move and accumulate across the food web?
In wastewater samples, branched isomers accounted for more than half of the total PFOS, whereas avian egg yolks from fish-eating double-crested cormorants were composed of approximately 90 percent linear PFOS. This pronounced shift in isomer composition across trophic levels suggests preferential bioaccumulation and/or retention of the linear isomer during trophic transfer. One plausible explanation is that PFOS interacts with biological macromolecules such as proteins and membrane lipids, and the linear isomer – due to its greater structural conformity with straight-chain lipid architectures – may exhibit stronger or more selective binding affinities. However, the mechanistic basis for this selective enrichment remains poorly understood, underscoring the need for further investigation into isomer-specific interactions governing PFOS bioaccumulation and maternal transfer.
Do you see any implications of this work for consumers, regulators, or industry – especially when it comes to how PFAS risks are assessed?
Our work suggests important implications for how PFAS risks are assessed by consumers, regulators, and industry. While current regulations typically focus on total PFOS, our findings highlight that distinguishing between linear and branched isomers is a critical first step toward more refined, science-based risk assessments, particularly if these isomers differ in environmental behavior or toxicity. Advancing this approach will require routine isomer-resolved monitoring, standardized analytical methods, and incorporation of isomer-specific toxicokinetics into risk assessment frameworks, including PBPK models. In parallel, improved analytical workflows, harmonized reporting, and interlaboratory validation are needed to support a transition from total PFAS metrics to isomer-specific evaluation. Complementary efforts – such as high-throughput screening, QSAR modeling, and machine learning – can help identify higher-risk isomers and link structure to health outcomes. Together, these advances can enable more accurate exposure assessment, inform targeted regulation and phase-outs, and support safer chemical design, ultimately strengthening PFAS management and protection of public health.
Looking ahead, what do you see as the most important directions for future research on PFAS – particularly when it comes to understanding isomers and their real-world impacts?
A key priority for PFAS research is to move toward an isomer-resolved framework that better reflects real-world exposures and risks. In the near term, this means encouraging regulatory agencies to adopt isomer-specific reporting in monitoring and compliance programs, alongside updated analytical methods, calibration standards, and quality assurance protocols that can reliably distinguish individual isomers. At the same time, integrating isomer-specific toxicokinetics into risk assessment – such as through refined health-based guidance values and physiologically based pharmacokinetic (PBPK) models – will be essential for improving the accuracy of exposure and hazard evaluations.
Equally important is expanding our understanding of the toxicity and biological behavior of individual PFAS isomers, since they may differ in bioaccumulation, persistence, and health effects. Future studies should broaden the range of environmental and biological matrices examined and extend beyond well-studied compounds like PFOS to include other PFAS classes. Ultimately, making isomer-specific measurements a routine part of environmental and biomonitoring efforts will provide a more complete and mechanistic understanding of PFAS fate, transport, and impacts – helping to inform more effective science-based policies and interventions.
Beyond this study, which of your ongoing PFAS-related projects are you most excited about right now, and why?
We are particularly excited about extending our analytical framework to achieve isomer-resolved characterization of a broader range of PFAS beyond PFOS, including shorter-chain and emerging compounds. This involves refining high-resolution separation techniques, such as advanced chromatography coupled with IMS to enable accurate quantification of individual isomers in complex biological matrices. Applying these methods to samples like human milk is especially important, as it provides a direct window into early-life exposure and can help elucidate isomer-specific transfer and bioaccumulation patterns.
In parallel, we are also interested in our ongoing work to elucidate the molecular mechanisms governing PFAS interactions with biological transporters. Specifically, we are investigating how structural differences among isomers influence binding affinities to serum proteins and membrane transporters, including those involved in translocation across the blood–brain barrier. Understanding these interactions at a mechanistic level will be critical for determining whether isomer-specific physicochemical properties translate into differences in tissue distribution, bioaccumulation, and ultimately toxicological outcomes.
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