Advances in Non-Targeted Analysis for PFAS in Environmental Matrices

You recently published a review of non-targeted analysis (NTA) methods for analyzing per- and polyfluoroalkyl substances (PFAS) in the environment (1). How do NTA methods compare to targeted methods in terms of sensitivity and detection limits for PFAS in environmental matrices?
A well-developed targeted method should provide lower detection limits for PFAS because it has been specifically designed to determine concentrations of those PFAS, whereas an NTA method is designed to be a broad sweep analysis. Mass spectrometers capable of performing NTA are getting more and more sensitive and so detection levels using NTA are falling. However, I would caution against comparing the two. Trying to perform NTA by chasing the detection limits capable of targeted analysis can weaken the power of NTA. There is a risk of it causing analysts to focus on optimizing performance for a few target analytes at low detection levels, which can impact the data quality for thousands of other analytes.

What are the most common PFAS classes detected through NTA in environmental samples, and how do they compare to targeted monitoring methods?
Perfluorosulfonic acids (PFSAs) and perfluorocarboxylic acids (PFCAs) are the most detected PFAS in NTA methods. These two classes contain the infamous perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) that underpin many targeted methods. NTA shows us that this is only the tip of the iceberg; there are thousands of PFAS in our environment and their concentrations and composition are highly variable, yet targeted methods only look for tens of PFAS. The most detected PFAS from NTA–those that are not in targeted methods–are chlorine-substituted PFAS such as chlorine substituted perfluorosulfonic acids (Cl-PFSAs) and chlorine substituted perfluorocarboxylic acids (Cl-PFCAs) However, the overwhelming variety of different PFAS detected shows that targeted methods alone are not capable of appropriately measuring the levels of PFAS in the environment.

David Megson from Manchester Metropolitan University in Manchester, UK,

How have the advances in liquid chromatography (LC) and negative electrospray ionization (ESI) techniques improved the detection of PFAS, and what further developments are needed in chromatographic technologies? The development of ultrahigh-performance liquid chromatography (UHPLC) has played an important role because it has helped to improve detection limits and chromatographic separation, which helps in the accurate quantification of more PFAS. Negative ESI works particularly well for many PFAS in targeted methods and is the most used ionization technique in NTA. However, it should be noted that the range of PFAS in the environment dictates the need for a variety of different methods; not all PFAS are the same! Some are positively charged, some are negatively charged, some are zwitterionic, some are neutral, and some are volatile. To provide the most comprehensive measurements of PFAS, we need combinations of gas chromatography (GC) and LC techniques operating in positive ionization (PI) and negative ionization (NI) modes.

What role do quality assurance and quality control (QA/QC) measures play in the reliability of NTA results, and how can they be strengthened to reduce false positives and negatives?
This is an area that is currently lacking in my opinion. We tend to apply QA/QC procedures from targeted methods to check the acceptability of NTA methods. They have different aims and so are not always appropriate. Blanks and standards are essential in both, but the limit of detection/limit of quantification (LOD/LOQ) measurements that are useful for targeted methods can be misleading for NTA as they are only reported for a few analytes. What we desperately need more of is reporting of rates of false positives and false negatives in NTA studies. Jacob et al.[2] have proposed a method for this; it would be great if it was more widely adopted.

With over 1000 PFAS identified across 382 different classes, what steps can be taken to ensure that NTA methods remain robust and capable of detecting rare or previously unknown PFAS compounds?
I think this is where the QA/QC procedures on false positives and negatives can help us to develop our methods to be as comprehensive as possible. I like that different research groups use different analytical methods to perform NTA as that helps increase the range of PFAS that we are detecting.

How do you address the subjective nature of many NTA workflows, and what approaches can be adopted to standardize these processes for greater consistency and reproducibility?
Thinking as an analytical chemist this is a bit of a nightmare, many highly subjective parameters in an NTA study mean different analysts can get different results from the same sample. Again, this is where the improved QA/QC procedures to monitor false positives and negatives can be used to optimize methods. This can mean that two separate analysts can each use a different method but return a result that is more accurate with improved precision.

What is your perspective on the role of biomonitoring in PFAS analysis, particularly concerning non-lethal blood sampling for humans and wildlife? What further biomonitoring studies are essential for understanding health risks?
It was always a frustration of mine when studies would kill animals to measure the level of pollution in them to establish if the pollution could be harming them. Advances in analytical instrumentation have meant lower detection limits, which means we now need fewer samples and opens up the possibility of performing NTA on tiny volumes of blood (we regularly use 50 µL blood samples). The majority of existing PFAS NTA studies are in water. Very few studies have been performed on humans and wildlife. The public is becoming increasingly concerned by the risks of PFAS exposure, but we currently have a poor understanding of exactly what PFAS we may have been exposed to. More NTA studies are needed in humans and wildlife to fill this knowledge gap.

Given that many studies focus on PFAS in environmental matrices rather than commercial products, how can NTA techniques be adapted to improve the detection of PFAS in industrial and consumer products?
This is another area that needs more research. There is a wide variety of different PFAS that are used in commercial products. One of the main challenges here lies in dealing with a range of unusual sample matrices. We have lots of experience working on environmental samples and so know how to process these effectively. Trying to comprehensively extract all PFAS from a new matrix is challenging, but our review (1] identified NTA studies have been used successfully on several commercial products. (3–8).

Given that NTA often focuses on freshwater, groundwater, soil, and sediments, how can methods be adapted or expanded to better analyze PFAS in marine environments and the atmosphere?
Mass spectrometry (MS) methods are largely transferable; the same analytical and data processing workflows can be used on any extract. Where most development is needed is in making sure the sample preparation method is appropriate for that matrix. Minimal sample preparation is preferable because any sample preparation technique has the potential to introduce contamination or remove analytes of interest. Our review (1) highlighted there have been NTA studies in both air (9–13) and seawater (14–16) so it is possible, and I hope there will be more research in both environments in the future.

What sample preparation techniques are most used for NTA of PFAS?
Solid phase extraction (SPE) based methods were the most widely used sample preparation methods and were adopted by 75% of studes included in our review [1]. There was a large range of different methods used with over 40 different types of sorbent combinations reported. Most studies used relatively simple preparation methods including; centrifuging, filtering dilute and shoot, sonication, vortexing. There was a surprising lack of automated methods, so this could be an interesting area for future development.

Can you discuss the impact of geographical bias in NTA studies on the overall understanding of PFAS contamination? What additional research is needed in underrepresented regions, such as South America and Africa?
This geographical bias, unfortunately, exists within most scientific disciplines. Funding for research in South America and Africa is much more limited compared to most of the global north. There are great scientists working in these regions, but with limited opportunities. It would be brilliant if there were more collaborative projects between scientists from more well-funded countries and those from understudied regions.

What are the main limitations of relying solely on NTA for PFAS detection, and how can integrated approaches combining both targeted and non-targeted methods help in more comprehensive environmental monitoring?
When compared against targeted methods NTA is more expensive and time-consuming, which is normally a barrier. I would propose using a combination of targeted methods and total PFAS methods (such as extractable organic fluorine or TOP assay) in routine analysis. These will help establish if targeted methods are accurately capturing all the PFAS in the sample. Where there is a large discrepancy, NTA can be used to fill in the blanks to help identify which PFAS are being missed.

Do you have any advice for scientists who are new to developing NTA techniques?
First, it’s great fun, so do give it a go! It can be a daunting task, so I would strongly suggest speaking with other experts before you start (including instrument manufacturers–not just other academics). I do encourage people not to chase the lowest detection limits and instead focus on optimizing a workflow that captures the range of chemistries you are looking for. Use a range of standards to optimize methods to achieve this goal. Finally, be wary of quoting concentrations for analytes you do not have standards for, as results will be semi-quantitative at best.

Any comments on the availability of reference standards?
This is a major barrier in NTA and restricts us from reaching confidence level one (the highest level of confidence on the Schymanski (17) scale. That said, with how good mass spectrometers are these days we can still reach a very high degree of confidence with many of our assignments. Tools like ultra high resolution mass spec, MS/MS experiments and ion mobility continue to give us multiple lines of evidence. I don’t think we will ever have reference standards for all 7 million + PFAS (18) but thankfully we have pretty awesome kit to help give us confidence in our assignments.

David Megson is a reader in chemistry and environmental forensics at Manchester Met University (UK) and Chemistry Matters (Canada). Heinvestigates sources of legacy and emerging persistent organic pollutants and monitors them in the environment using advanced analytical techniques including multidimensional chromatography and high-resolution mass spectrometry. Much of Megson’s research has an environmental forensics aspect, which involves identifying the sources of contamination, transport pathways, and the magnitude of pollutant exposure. He regularly contributes as a recognized international expert to witness reports for large (>$100 million) litigation cases involving environmental pollution in North America. His current research group includes six PhD students and two postdoctoral researchers. He has attracted >£4.5 million of research funding since he was appointed a lecturer in 2016. Recently, he has been investigating the scale of PFAS pollution in the UK and has published papers investigating releases from industrial sites [19] and wastewater treatment plants [20].

References

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2. Jacob, P.; et al. Evaluation, Optimization, and Application of Three Independent Suspect Screening Workflows for the Characterization of PFASs in Water. Environ. Sci. Process. Impacts 2021, 23(10), 1554–1565. DOI: https://doi.org/10.1039/D1EM00286D

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9. Ghorbani Gorji, S.; et al. New PFASs Identified in AFFF-Impacted Groundwater by Passive Sampling and Nontarget Analysis. Environ. Sci. Technol. 2024, 58(3), 1690–1699. DOI: 10.1021/acs.est.3c06591

10. Mok, S.; et al. Target and Non-target Analyses of Neutral Per- and Polyfluoroalkyl Substances from Fluorochemical Industries Using GC-MS/MS and GC-TOF: Insights on Their Environmental Fate. Environ. Int. 2023, 182, 108311. DOI: https://doi.org/10.1016/j.envint.2023.108311

11. Qiao, B.; et al. Nontarget Screening and Fate of Emerging Per- and Polyfluoroalkyl Substances in Wastewater Treatment Plants in Tianjin, China. Environ. Sci. Technol. 2023, 57(48), 20127–20137. DOI: 10.1021/acs.est.3c03997

12. Yu, N.; et al. Non-Target and Suspect Screening of Per- and Polyfluoroalkyl Substances in Airborne Particulate Matter in China. Environ. Sci. Technol. 2018, 52(15), 8205–8214. DOI: 10.1021/acs.est.8b02492

13. Yu, N.; et al. Nontarget Discovery of Per- and Polyfluoroalkyl Substances in Atmospheric Particulate Matter and Gaseous Phase Using Cryogenic Air Sampler. Environ. Sci. Technol. 2020, 54(6), 3103–3113. DOI: 0.1021/acs.est.9b05457

14. Chen, H.; et al. Occurrence and Distribution of Per- and Polyfluoroalkyl Substances in Tianjin, China: The Contribution of Emerging and Unknown Analogues. Environ. Sci. Technol. 2020, 54(22), 14254–14264. DOI: https://doi.org/10.1021/acs.est.0c00934

15. Dunn, M.; et al. Unregulated Active and Closed Textile Mills Represent a Significant Vector of PFAS Contamination into Coastal Rivers. ACS ES&T Water 2024, 4(1), 114–124. DOI: https://doi.org/10.1021/acsestwater.3c00439

16. Wang, Q.; et al. Legacy and Emerging Per- and Polyfluoroalkyl Substances in a Subtropical Marine Food Web: Suspect Screening, Isomer Profile, and Identification of Analytical Interference. Environ. Sci. Technol. 2023, 57(22), 8355–8364. DOI: https://doi.org/10.1021/acs.est.3c00374

17. Schymanski, E. L.; et al. Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence. Environ. Sci. Technol. 2014, 48(4), 2097–2098. DOI: https://doi.org/10.1021/es5002105

18. Schymanski, E. L.; et al. Per- and Polyfluoroalkyl Substances (PFAS) in PubChem: 7 Million and Growing. Environ. Sci. Technol. 2023, 57(44), 16918–16928. DOI: https://doi.org/10.1021/acs.est.3c04855

19. Megson, D.; et al. Non-targeted Analysis Reveals Hundreds of Per- and Polyfluoroalkyl Substances (PFAS) in UK Freshwater in the Vicinity of a Fluorochemical Plant. Chemosphere 2024, 367, 143645. DOI: https://doi.org/10.1016/j.chemosphere.2024.143645

20. Neill, P.; Megson, D. Landfill Leachate Treatment Process is Transforming and Releasing Banned Per- and Polyfluoroalkyl Substances to UK Water. Front. Water 2024, 6. DOI: https://doi.org/10.3389/frwa.202