The Benefits of Stacked SPE Cartridges for PFAS Analysis

Per- and polyfluorinated alkyl substances (PFAS) are environmentally persistent, water-soluble compounds widely used in industrial and firefighting applications. Their bioaccumulative nature and links to adverse health effects have driven the development of increasingly sensitive and robust analytical methods. Initial EPA methods focused on drinking water, while Method 533 introduced improved detection of short-chain and precursor PFAS using weak anion exchange (WAX) solid-phase extraction (SPE) and isotope dilution. For non‑potable matrices, laboratories developed 537MOD methods, later standardized by the Department of Defense (DoD) through Quality Systems Manual (QSM) table B-15, which mandated the use of graphitized carbon black (GCB) to mitigate cholic acid interference. However, traditional GCB clean-up methods introduced workflow inefficiencies and risked long-chain PFAS loss. In response, a novel stacked SPE cartridge combining WAX and GCB was introduced, enabling simultaneous extraction and cleanup while maintaining compliance with table B-15. This article discusses their utility in food safety and total organic fluorine workflows, offering enhanced sensitivity, reproducibility, and operational efficiency.

Per- and polyfluorinated alkyl substances (PFAS) are fluorinated carbon chains of various lengths with different functional groups, such as sulfonic or carboxylic acids. Compounds with lengths of less than 22 carbons are the most widely studied, but this is primarily due to commercial availability of analytical standards and isotopic standards. PFAS are a class of chemicals historically and currently used in various industrial processes, such as the manufacturing of paper products, textiles, and food packaging. They are also the primary component in aqueous film forming foam (AFFF), which is used to suppress jet fuel fires at airports and military bases. PFAS compounds are extremely mobile in the environment due to their unique structure and the most studied PFAS are very water soluble. Depending on the chain length of the PFAS, they can accumulate in tissue and can be biomagnified, which creates a bigger problem with organisms at the top of the food chain (such as large carnivores, as well as people). There are many known effects on endocrine function, immune system function, and fetal development, as well as links to several forms of cancer. The most troubling part might be their reputation as forever chemicals, substances that persist indefinitely. These compounds are exceptionally stable and do not degrade under normal environmental conditions.

These chemicals have been around and in use since the 1940s, but it wasn’t until the early 2000s that there were any published methods. These first methods were plagued by significant limitations in both scope and sensitivity. Very few analytical standards were available, and the mass spectrometry (MS) detectors of that era were much more influenced by matrix interferences. Despite these limitations, alot of research was being done on PFAS as an emerging persistent organic pollutant (POP), and there were many analytical methods developed, although most used gas chromatography (GC) (1–3).

The first official analytical method was developed by the Environmental Protection Agency (EPA) in 2009 to monitor PFAS in drinking water. EPA Method 537 allowed for the quantitation of 14 PFAS compounds that were extracted from drinking water using a styrene divinylbenzene (SDVB) sorbent, allowing for concentration of the PFAS and removing matrix compounds to increase sensitivity. This method was not very selective to many shorter chain PFAS compounds because the solid-phase extraction (SPE) sorbent did not have good chemical interaction with smaller or more polar analytes. In addition, SDVB would also interact with many other organic compounds that would have a negative impact on the sensitivity of the MS detector. Although Method 537 was validated for the detection of PFAS in drinking water, cleaner matrices allowed for the inclusion of several PFAS compounds in the Third Unregulated Contaminant Monitoring Rule (UCMR 3) study of public water systems in the United States. Method 537 was revised in 2019 to Method 537.1, with the addition of four replacement small PFAS chemicals that were being used as alternatives to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), which had been phased out in the early 2000s because of increasing awareness of health concerns.

In 2019, the EPA released Method 533, a newer analytical method for PFAS in drinking water. This method had some significant changes to the 537 predecessors, including:

• Removal of four long-chain analytes (that had a history of non-detect in drinking water).
• Addition of eight short-chain PFAS (C<8) as well as three precursor fluorotelomer sulfonic acid (FTS) compounds (with a total of 25 analytes).
• SPE by weak anion exchange (WAX), which is much more selective to anionic PFAS compounds (most targeted PFAS are weak sulfonic or carboxylic acids).
• Isotope dilution for extracted internal standard (EIS) compounds to provide some normalization of SPE loss and matrix effects on analyte quantitation.

The use of the weak anion exchange sorbent was the biggest modification, as this chemistry provided improved extraction recoveries for most of the newer replacement and precursor PFAS. In fact, most of the analytical methods that have come after Method 533 have also used WAX chemistry for SPE, including methods for food and air matrices (4–7).

By 2019, there were two official methods for PFAS in drinking water, but none for PFAS in other environmental matrices, such as wastewater and soil. There were “user defined” methods for these matrices that were developed independently by many commercial laboratories designated as “537MOD.” This nomenclature was deceiving, as the only connection to Method 537 was that these methods were used to analyze PFAS; this naming provided a degree of legitimacy for customers that needed to determine PFAS in non‑drinking water matrices.

Most of these 537MOD methods had a degree of similarity. They all used WAX SPE to remove matrix and concentrate compounds of interest, and all used isotope dilution to improve the accuracy, sensitivity, and robustness. However, since no consistent or standard quality control (QC) metrics existed, it was difficult to compare results from different laboratoriess using their own 537MOD methods. This changed in 2019 when the Department of Defense (DoD) published the first comprehensive QC guidelines for PFAS analysis. These guidelines were part of Quality Systems Manuals (QSM) version 5.3 and were codified in table B-15. Labs that were accredited to these QC parameters could now theoretically have comparable data, which allowed for better regulatory and statutory decisions regarding PFAS contamination.

One of the unique criteria that the DoD included in table B-15 was a requirement to use graphitized carbon black (GCB) as part of the sample cleanup for PFAS workflows. This requirement was born out of concern that samples from matrices with high organic content, such as wastewater, soils, sediments, and tissues, would contain cholic acids, which are generated from the breakdown of organic matter. Cholic acids, particularly species like taurodeoxycholic acid (TDCA), can coelute with PFOS, and will generate a daughter ion in the first fragmentation of MS/MS analysis with the same mass‑to-charge (m/z) ratio, thus leading to high bias in quantifying this compound. GCB has a strong affinity for cholic acids, and treating the sample with this sorbent will reduce the amount of cholic acids in the sample, which can lead to more accurate results for PFOS.

While this requirement added improved data integrity to these methods, it created several problems within the workflow. Table B-15 reflected the requirement to use GCB, but did not provide any details or guidance on how to apply or expose the sample to this sorbent. Historically, GCB was applied to the sample as either loose sorbent (in a process called dSPE, or dispersive solid-phase extraction), or the sample was passed through a cartridge containing GCB, which is basically SPE using GCB as the sorbent. Both techniques had downsides. The loose GCB produced problems with powder mess, which is extremely electrostatic and clings to any surface. In addition, using loose GCB required several other steps in the workflow, including sonication to increase interaction between cholic acid interferences and the sorbent, centrifuging to separate the loose GCB from the treated sample, and filtration to remove any remaining GCB particles that could clog the liquid chromatographic (LC) column or the MS source. GCB in a cartridge contributed more cost to the method, as well as adding more time and decreasing productivity. Both techniques were also plagued by the potential for loss of longer chain PFAS compounds if the sample was exposed to the GCB for too long.

In 2020, a collaboration was formed to develop a new SPE format that would simplify adherence to the table B-15 requirement to use GCB as part of the sample preparation. This new format would layer or stack WAX media with the GCB in a single cartridge, and the SPE would then be both an extraction of the PFAS as well as a removal of the cholic acid interferences. This new cartridge was designed and optimized for PFAS workflows requiring table B-15 compliance (at that time this was all 537MOD methods that were DoD compliant). As this was such a novel solution to the requirement of GCB, there was concern from the DoD that the stacked SPE format would provide equivalent results to the traditional use of loose GCB or separate GCB cartridges. As a result, the DoD required a technical note with data to support that these cartridges would meet all of the QC parameters established by table B-15. This data is provided in Tables I and II.

Initially there were two sorbent formats of stacked SPE for PFAS that were commercially available: 200 mg of WAX stacked on top of 50 mg of GCB, or 500 mg of WAX stacked on 50 mg of carbon (depending on the WAX sorbent mass that was already being used by a commercial lab running a 537MOD workflow) (8). These were the only stacked cartridges available for PFAS until 2021, when the EPA released the first draft of their new 1633 Method. This method was specifically developed for the analysis of PFAS in non-drinking water matrices (aqueous matrices) such as wastewater, surface water, or ground water, or solid matrices such as soils, sediments, biosolids, leachates, and tissue. This new method was developed as a collaborative effort between the EPA and the DoD and included the table B-15 requirement to treat the sample with GCB. The first draft of Method 1633 included language indicating that the single-laboratory validation (SLV)of this method had used loose GCB, and that cartridges containing GCB, including the stacked format, would only be allowed following the generation of final QC specifications during the multi‑laboratory validation (MLV)process. In December 2022, following the analysis of MLV data for wastewater and the calculation of final QC specifications for this matrix, the EPA released the 3^rd Draft of Method 1633, which allowed for the use of stacked WAX/GCB cartridges on wastewater samples, and the 4^th Draft (January 2024) allowed for the use of these formats for all other sample matrices.

EPA Method 1633 also had the additional requirement that the sequence of WAX and GCB be dependent on the type of sample matrices. For aqueous matrices, the sequence for treating the sample was SPE with WAX followed by treating with GCB, so the cartridges had the WAX stacked on top of the GCB. For solid matrices, the sample was first exposed to GCB and then extracted by the WAX phase, so the cartridges would have the GCB stacked on top of the WAX. As a result, there was commercial development of the reverse stack SPE cartridges, in which the GCB was layered on top of the WAX sorbent. In November 2024, there were two technical notes published outlining the performance of stacked SPE cartridges in standard stack and reverse stacked formats meeting all method QC requirements for EPA Method 1633 (Figures 1, 2, and 3) (9,10).

Since 2024, there have been more companies that adapted this SPE format for Method 1633 using the original 200 mg WAX and 50 mg GCB sorbent masses. However, because of their long experience with these SPE cartridges, there were some SPE suppliers that recognized that 50 mg of GCB could be problematic in samples with a lower organic background. In these cases, exposure to GCB could lead to lower recoveries for long-chain PFAS (C>8), as these compounds had much greater interaction with GCB. As a result, several other stacked SPE formats using only 10 mg of GCB were commercialized. This amount of GCB still effectively removed cholic acid and also increased productivity by decreasing the time spent extracting each sample due to 5× less GCB sorbent to elute through.

While the use of GCB for PFAS sample preparation was initially tied
to projects using Method 1633 or 537MOD methods, there has been a lot of work in other PFAS workflows in which these stacked SPE cartridges would provide additional benefits. Several papers have been published using stacked SPE format in food matrices (Nestle 2023 and Institute of Food Chemistry 2024) to streamline sample preparation as well as to improve sensitivity and robustness (11,12). Stacked SPE was also used in a study looking at extractable organic fluorine (EOF) using SPE vs. absorbable organic fluorine (AOF) using activated carbon in determining total organic fluorine, with the stacked SPE format providing improved recoveries and lower LOD and LOQ being attained (13). It is anticipated that there will be continued development and refinement of these novel SPE formats for PFAS, especially as methods require more sensitivity and robustness, and higher productivity for commercial labs.

References

(1) U.S. Environmental Protection Agency. An Introduction to Per- and Polyfluoroalkyl Substances (PFAS). Presentation by Tim Watkins, Center for Public Health and Environmental Assessment. September 29–30, 2021. https://www.epa.gov/system/files/documents/2021-09/2_pfas-introduction_watkins.pdf

(2) U.S. Environmental Protection Agency. Multi-Industry Per- and Polyfluoroalkyl Substances (PFAS) Study – Preliminary Report. Office of Water, EPA-821-R-21-004, September 2021. https://www.epa.gov/system/files/documents/2021-09/multi-industry-pfas-study_preliminary-2021-report_508_2021.09.08.pdf

(3) Interstate Technology and Regulatory Council (ITRC). History and Use of Per- and Polyfluoroalkyl Substances (PFAS) Fact Sheet. September 2023. https://pfas-1.itrcweb.org/wp-content/uploads/2023/10/HistoryandUse_PFAS_Fact-Sheet_Sept2023_final.pdf

(4) Shoemaker, J. A.; Grimmett, P. E.; Boutin, B. K. Method 537, Revision 1.1: Determination of Selected Perfluorinated Alkyl Acids in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS). U.S. Environmental Protection Agency, Office of Research and Development, 2009.

(5) Shoemaker, J. A.; Tettenhorst, D. R. Method 537.1, Revision 2.0: Determination of Selected Per- and Polyfluorinated Alkyl Substances in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS). EPA/600/R-20/006. U.S. Environmental Protection Agency, Office of Research and Development, 2020.

(6) U.S. Environmental Protection Agency. Method 533: Determination of Per- and Polyfluoroalkyl Substances in Drinking Water by Isotope Dilution Anion Exchange Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry. EPA/815-B-19-020, 2019.

(7) U.S. Environmental Protection Agency. Comparing EPA Analytical Methods for PFAS in Drinking Water, 2024. https://www.epa.gov/dwanalyticalmethods/comparing-epa-analytical-methods-pfas-drinking-water

(8) Jack, R.; Lodge, S.; Robinson, A.; Johnson, T. Comparison of PFAS Sample Preparation Between WAX + Dispersive GCB and WAX/GCB Cartridges from Water and Soil Extracts Per Draft EPA 1633. The Column 2022, 18 (11), 28–33.

(9) Shimizu, M.; Butt, C. M.; Bassignani, P.; et al. Comparison of PFAS Extraction Efficiency Between SPE + dSPE versus Single Cartridge per EPA Method 1633. Phenomenex Tech Note. https://www.phenomenex.com/documents/2024/07/19/16/57/comparison-of-pfas-extraction-efficiency-between-spe--dspe-versus-single-cartridge-per-epa-method-16

(10) Shimizu, M.; Butt, C. M.; Bassignani, P.; et al. Use of a Simplified Extraction Method Using a Stacked SPE for Soil Extracts for EPA Method 1633. Phenomenex Tech Note. https://www.phenomenex.com/documents/2024/07/19/16/57/use-of-a-simplified-extraction-method-using-a-stacked-spe-for-soil-extracts-for-epa-method-1633

(11) Theurillat, X.; Mujahid, C.; Eriksen, B.; et al. Quantitation of PFAS in Food with Parts per Trillion Levels of Sensitivity. Phenomenex Technical Note, 2023.

(12) Beadle, A. An Analytical Toolbox for Tackling PFAS in Food. Technology Networks 2024. https://www.technologynetworks.com/applied-sciences/articles/an-analytical-toolbox-for-tackling-pfas-in-food-394163 (accessed 2025-08-14).

(13) Forster, A.; Zhang, Y.; Westerman, D.; et al. Optimized Total Organic Fluorine Methods Using Strata PFAS SPE Cartridges for a More Comprehensive Measurement of PFAS in Environmental Samples. Phenomenex Technical Note, 2023.

Sam Lodge is the Phenomenex senior business development manager for the environmental industry. He has been at Phenomenex for over 30 years in various positions, including key account manager and senior international sales manager, and is a product expert in SPE and HPLC chemistries. Over the past 8 years, he has worked extensively with commercial and government labs in the development and support of PFAS analytical methods, including the development and validation of EPA 533, the MLV for EPA 1633, and many internal (MOD) methods. He received a Bachelor of Science from the University of California, Santa Barbara. In his spare time, he enjoys fishing and surfing (but not at the same time!)