If you’ve been following our recent blogs, you’ve probably seen quite a bit on how per- and polyfluoroalkyl substances (PFAS) are shaking up both the food and environmental industry.

Even if you’ve not been following our blogs, you’ve probably seen a lot of media coverage like this one about how PFAS is affecting water supplies. Or perhaps you’ve caught the new Mark Ruffalo movie, Dark Waters? PFAS is hard to avoid—both in the media and in the world around us.

Yet in the world of analytical testing, the challenges to understanding and measuring PFAS may be less publicized, but not less important. But can LC-MS/MS technology lead the way in the analytical science, both for target (known) molecules and also for those molecules yet to be discovered? Let’s discuss this in more detail here.

One thing is for sure, the number of newly discovered PFAS is expanding. There are more than 4,700 known PFAS with more than 3,000 that may be environmentally significant.¹ With emerging PFAS such as precursors and GenX compounds, there’s still much work needed to identify and understand the impact of these chemicals. To give you some context, some studies have looked at characterizing PFAS in fire-fighting foams (aqueous film-forming foam; AFFF).^2-3 A key takeaway from the studies I cite here are that there are more than 40 classes of PFAS with multiple chain length each in the commercial mixtures of fire-fighting foams. Some of those can degrade to become perfluorinated carboxylates and sulfonates compounds, which are of high regulatory interest. 

Another challenge when identifying PFAS is recognizing the various intermediates along with the transformation to perfluoroalkyl end products in the environment. This is important because the end product depends on the chain length of the starting material. A single polyfluoroalkyl parent could generate a mixture of related intermediate polyfluoroalkyl transformation products and terminal recalcitrant PFAAs.^3-4 

Figure 1: PFAS family tree. Image source: https://www.scientificamerican.com/article/the-fluorine-detectives/

Analytical science is complicated, with a multitude of techniques available. Choosing the right solution for a particular experiment is important to give the best chance of getting the “right” answer to a question. Mass spectrometry, over time, has become one such technique that has become quite well accepted in many applications, and PFAS analysis is one such area. LC-MS/MS, in particular, provides a highly sensitive, specific and selective way to aid in both the targeted analysis of PFAS compounds and also the identification of “suspect” or “unknown” PFAS molecules by measuring the chemical molecular weight via a mass to charge ratio (m/z). But before I go on, I’d like to make it clear that there is no single analytical technique as a solution. It all comes down to using the right tool for the right job, of which mass spectrometry plays a significant part in seeing the “bigger picture.”

 Let’s take a look at some common techniques used to discover PFAS.

Figure 2: Selectivity and inclusivity associated with total organofluoride methods. Image source: Mcdonough, Carrie A., et al., 2019, Current Opinion in Environmental Science & Health

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Typically, PFAS is analyzed by LC-MS/MS methods, such as EPA Method 537, ISO 25101, DIN 38407-42, ASTM D7979, or ASTM D7968. LC-MS/MS can be an advantage in terms of sensitivity and selectivity, especially with targeted screening using a triple quadrupole MSMS instrument with a multiple reaction monitoring approach (MRM). This technique can help you accurately detect and quantify lower concentrations levels of PFAS. While, perfluororinated substances do not react with anything, making it a “forever chemical,” polyfluorinateds could oxidize to perfluorinated persistent end products.^2-3

From an untargeted analysis perspective, this can cause a big challenge. If you don’t know what to look for—you are going to have a problem finding it. And herein lies the problem with regards to using targeted LC-MS/MS methods when discovering new PFAS. You have to consider other techniques, which in the mass spectrometry domain, could by the use of high-resolution accurate mass instruments such a hybrid quadrupole time of flight (QTOF) to aid in the identification of both suspect and “unknowns” through the increased specificity provided by such instrumentation. See later in this blog to learn more about the benefits of accurate mass for PFAS research.

Total oxidizable precursors (TOP) assay

TOP assay is a hydroxyl radical-based oxidation reaction. Precursors are transformed into perfluoroalkyl acids (PFAAs). This technique offers a view of PFAS being present in specific samples, but it doesn’t match the selectivity of identification provided by mass spectrometry.

The technique is complementary to LC-MS/MS as it helps give an indication of the total amount of perflourinated species in a sample, but not the type. Using TOP assay, you can see how much matter gets oxidized into perfluorinated end-products. However, the selectivity of TOP assay is limited to the oxidized compounds to form LC amenable hydroxyl radical resistant PFAS. Simply put, any precursors that oxidize to untargeted PFAS can be missed. What’s more, low and variable recoveries could result in false-negative.⁵ So in some ways, it’s safe to say TOP assay is only effective if you have at least a targeted screening LC-MS/MS to measure the concentration of perfluorinated species generated.

Figure 2 also shows you as you move out further from the center, the techniques are less specific. Those techniques focus more on total organic fluorine, organic fluorine with CF3 groups or even more broadly total fluorine. So, you lose some important information in terms of the behavior of the organic compounds.

High-resolution mass spectrometry (HRMS)

As discussed before, targeted analysis of PFAS with mass spectrometry is generally the domain of a highly sensitive and selective triple quadrupole LC-MS/MS. LC-MS/MS also seems to be a method used in research by the United States, the Food and Drug Safety Administration, which published a scientifically validated method using this technique to analyze food-related samples. Additionally, a well-established method endorsed by the Environmental Protection Agency (EPA) focusing on water testing, also recommends two validated methods that use LC-MS/MS techniques—EPA Method 537.1 and  Method 533 to analyze 29 PFAS in drinking water. This includes the replacement compounds in the market now HFPO-DA (a component of the GenX processing aid technology) that is generating a lot of attention due to the contamination in Cape River, North Carolina.

The diverse nature of PFAS compounds makes analytical testing extremely complex. PFAS dark matter could transform in the environment outside the scope of current regulatory interest. What this means is there is a lot of unknown dark matter that is often not captured by routine analytical methods. This is where accurate mass technology, HRMS, more specifically, can play a critical role in aiding in the identification of PFAS-related species, both in food and environmental samples. To learn more, watch this webinar with Professor Christopher Higgins on his groundbreaking research to analyze PFAS using the SCIEX X500R QTOF accurate mass LC-MS/MS technology.

Another interesting, accurate mass instrumentation point to mention involves selectivity. The selectivity of the analytical data is increased through improved mass resolution (typically greater than 30,000 FWHM), which allows an “accurate mass” measurement for a particular analyte to be made. Measurements are typically to 4 decimal places, which allows a potential elemental formula to be postulated. This is hugely beneficial when doing “suspect” compound identification as it will enable the analyst to “hone in” on a particular molecule structure with usual accuracies in the region of 1ppm from the actual “theoretical” mass of a molecule. Couple this with having accurate mass MSMS, which provides the fine detail of a molecule structure, new entities that have not previously been targeted can potentially be identified with greater ease. Using accurate mass technology, particularly with MSMS information, allows you to be more confident in your analytical answers—a huge benefit when dealing with the multitude of PFAS molecules in the environment.

In a nutshell

Accurate mass MS/MS technology gives researchers enhanced mass spectrometry power to address challenging analytical questions. By applying the advanced features and software workflows of today’s QTOF instrumentation, researchers have access to screening, selectivity and data processing capabilities to meet the modern needs of PFAS testing. QTOF instruments like the SCIEX X500R offers you:

• High-quality, accurate mass MSMS spectra for enhanced information
• Fast scanning capability to be able to deal with fast LC separations, particularly important when dealing with complex samples and matrices
• No tradeoff of mass resolution, sensitivity or dynamics range even at 100Hz data acquisition rates
• A complete digital record of a single sample allowing you to “mine” the data as you see fit
• Compound confirmation against newly expanded PFAS MS/MS spectral library
• Targeted screening for “legacy” PFAS compounds (i.e., PFOS, PFOA) using MRM-HR, full-scan MRM-HR for SCIEX PFAS MS/MS Library v2.0 that contains 252 PFAS compounds covering negative, positive and zwitterionic compound classes

With accurate mass MSMS, you can benefit from advanced analytical capabilities to experience outstanding performance and streamlined workflows for rapid PFAS detection and identification.  So, to answer the question, what is the ideal analytical technique for discovering new PFAS? I recommend that your lab consider QTOF technology to your analytical instrumentation armory as one potential and complementary solution to all the techniques available. It will certainly help with looking at the bigger picture associated with complex PFAS analysis.  Click here to download the X500R QTOF PFAS content pack to learn more about how the instrument can help capture PFAS dark matter.

If your lab is more focused on monitoring regulatory limits, read our blog on routine, targeted PFAS testing to learn about one way to detect low levels of the substance effectively.

References

1. Wang, Zhanyun, et al. “A Never-Ending Story of Per- and Polyfluoroalkyl Substances (PFASs)?” Environmental Science & Technology, vol. 51, no. 5, 2017, pp. 2508–2518., doi:10.1021/acs.est.6b04806.
2. Barzen-Hanson, Krista A., et al. “Discovery of 40 Classes of Per- and Polyfluoroalkyl Substances in Historical Aqueous Film-Forming Foams (AFFFs) and AFFF-Impacted Groundwater.” Environmental Science & Technology, vol. 51, no. 4, Feb. 2017, pp. 2047–2057., doi:10.1021/acs.est.6b05843.
3. D’Agostino, Lisa A., and Scott A. Mabury. “Identification of Novel Fluorinated Surfactants in Aqueous Film Forming Foams and Commercial Surfactant Concentrates.” Environmental Science & Technology, vol. 48, no. 1, 2013, pp. 121–129., doi:10.1021/es403729e.
4. Harding-Marjanovic, Katie C., et al. “Aerobic Biotransformation of Fluorotelomer Thioether Amido Sulfonate (Lodyne) in AFFF-Amended Microcosms.” Environmental Science & Technology, vol. 49, no. 13, 2015, pp. 7666–7674., doi:10.1021/acs.est.5b01219.
5. Avendaño, Sandra Mejia, and Jinxia Liu. “Production of PFOS from Aerobic Soil Biotransformation of Two Perfluoroalkyl Sulfonamide Derivatives.” Chemosphere, vol. 119, 2015, pp. 1084–1090., doi:10.1016/j.chemosphere.2014.09.059.
6. Houtz, Erika F., and David L. Sedlak. “Oxidative Conversion as a Means of Detecting Precursors to Perfluoroalkyl Acids in Urban Runoff.” Environmental Science & Technology, vol. 46, no. 17, 2012, pp. 9342–9349., doi:10.1021/es302274g.
7. Houtz, Erika F., et al. “Persistence of Perfluoroalkyl Acid Precursors in AFFF-Impacted Groundwater and Soil.” Environmental Science & Technology, vol. 47, no. 15, June 2013, pp. 8187–8195., doi:10.1021/es4018877.
8. Dimzon, Ian Ken, et al. “High Resolution Mass Spectrometry of Polyfluorinated Polyether-Based Formulation.” Journal of The American Society for Mass Spectrometry, vol. 27, no. 2, 2015, pp. 309–318., doi:10.1007/s13361-015-1269-9.
9. Figure 2: Mcdonough, Carrie A., et al. “Measuring Total PFASs in Water: The Tradeoff between Selectivity and Inclusivity.” Current Opinion in Environmental Science & Health, vol. 7, 2019, pp. 13–18., doi:10.1016/j.coesh.2018.08.005.

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