Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Accordance with EPA 1633 Part 3: Analysis of Soil and Tissue

US EPA Method 1633 has become the foundational method for analysis of PFAS in non-potable water matrices, soils, biosolids, and tissues in the United States. The method consists of sample preparation using weak anion exchange (WAX) solid phase extraction (SPE) with graphitized carbon black (GCB) clean up. This application note is the third in a series demonstrating a comprehensive solution for performing the EPA 1633 methodology. The focus of this note is preparation and analysis of soil and fish tissue samples utilizing a bilayer dual-phase SPE cartridge and LC-MS/MS method on an ACQUITY™ Premier BSM FTN LC System coupled to a Xevo™ TQ Absolute Tandem Quadrupole Mass Spectrometer.

Benefits

• An end-to-end workflow is presented for PFAS analysis in soil and fish tissue samples following the EPA 1633 procedure
• A bilayer dual-phase SPE cartridge containing WAX and GCB was utilized to reduce the debris and hazards of working with dispersive GCB as well as further reducing sample preparation time
• Performance criteria of EPA 1633 are met for solids and tissues demonstrating equivalency of the workflow used
•  Performance of the workflow is demonstrated by easily passing qualifications of a Waters™ ERA certified reference material

US EPA Method 1633 was first introduced in August 2021 to become the foundational method for analysis of PFAS in non-potable water matrices, soils, biosolids, and tissues.¹ EPA 1633 was finalized in January 2024 and is multi-lab validated for each type of sample matrix included in the method.² The method covers 40 PFAS and utilizes isotope dilution calibration and quantitation. Required sample preparation differs slightly depending on sample type, but all sample types utilize solid phase extraction (SPE) on a weak anion exchange (WAX) cartridge in combination with graphitized carbon black (GCB) clean up. EPA 1633 was created to support sample analysis for the Clean Water Act (CWA) and Department of Defense (DoD) monitoring and remediation, but it covers such a wide range of matrices and compounds that its applicability is expected to be widespread.

This is the third in a series of application notes addressing sample preparation, analysis, and method performance of EPA 1633 using a comprehensive workflow of Waters technologies. This application note will focus on the preparation of authentic soil and tissue samples with analysis utilizing the LC-MS/MS method established in Part 1 on an ACQUITY Premier BSM FTN UPLC System coupled to a Xevo TQ Absolute Mass Spectrometer.³ The use of a combined WAX and GCB sample extraction and cleanup workflow is demonstrated on soil and fish tissue.

Sample Preparation

Samples discussed in this application note include soil and fish tissue. Salmon was used as the fish tissue matrix studied and was sourced at a local market. The fish tissue was homogenized using a blender before subsampling. The soil was a custom soil reference material created by the ERA that is similar to the PFAS in Soil CRM currently offered (Item number 603). It contained the 40 EPA 1633 PFAS at a known concentration. Samples were frozen until sample analysis according to EPA 1633 guidelines and holding times.

Oasis GCB/WAX for PFAS dual-phase SPE cartridges (p/n: 186011112) containing both WAX and GCB sorbents required for sample cleanup were used instead of using dispersive GCB. For soil and tissue analysis, the GCB is packed on top of the WAX sorbent to replicate the EPA 1633 where the GCB is used to clean the sample prior to WAX SPE.

Full sample preparation details are listed in Figures 1 and 2 and are adapted directly from the EPA 1633 method. Figure 1 details the two different extraction procedures used for soils/solids and tissues. Figure 2 details the SPE procedure used for all sample types. The dispersive GCB step was combined into the SPE cartridge, as described previously, providing the convenience of minimizing complications from using loose GCB material and reducing the number of steps during sample preparation without compromising the method.

Soil samples were spiked with 0.25–2 ng/g (sample concentration equivalent) of the required extracted internal standard (EIS) prior to extraction and 0.25–1.0 ng/g (sample concentration equivalent) of the required non-extracted internal standard (NIS) after extraction. Tissue samples were spiked with 0.625–5 ng/g (sample concentration equivalent) of the required extracted internal standard (EIS) prior to extraction and 0.625–2.5 ng/g (sample concentration equivalent) of the required non-extracted internal standard (NIS) after extraction. Individual concentrations vary dependent on the concentration of each component in the Wellington standard mixes. The calibration curve range for each analyte (in vial equivalent) is listed in Appendix Table 2 and was determined from the data acquired and presented in Part 1 of this application note series.³ All standards were obtained as mixes from Wellington Laboratories.

Recovery in Soil and Tissue Samples

EPA 1633 is a performance-based method that allows modifications as long as the performance criteria outlined in the method are all met. The only modification presented in this work was to use a bilayer dual-phase SPE cartridge that combines the otherwise dispersive GCB clean up step into the WAX SPE cartridge. GCB is difficult to work with and accurately measure, therefore utilizing a bilayer cartridge eliminates the untidy dispersive GCB step. More importantly, combining the GCB cleanup step into the SPE extraction saves valuable time in the laboratory during the sample preparation process. Additionally, less preparation steps allow for fewer opportunities for introduction of unintended PFAS sample contamination. For this work, a cartridge with the GCB stacked on top of the WAX was utilized to replicate the workflow of EPA 1633 where the GCB clean up step occurs before loading the sample onto the WAX cartridge.

One of the important performance criteria that must be established in order to prove equivalence of this approach is the target analyte (natives) and extracted internal standard (EIS) recovery acceptance limits (See Table 7 within that document).¹ The individual recovery performance of the bilayer dual-phase SPE cartridge for soil and fish tissue are listed for each EIS in Table 1. The data reported in Table 1 is the average recovery and %RSD for 3 replicate extractions of each matrix type. The mean recovery of all EIS among the soil and tissue samples extracted was 81% and 85%, respectively, with mean RSDs of 2.8% and 9.2% for soil and tissue, respectively.

Figure 3 directly compares the average recovery for the EIS in each sample type with the allowable recoveries in EPA 1633 Table 7. All PFAS analyzed in this study in both soil and fish tissue, were easily within the recovery acceptance limits for each compound, and in all cases were significantly above the minimum recovery level, demonstrating that the bilayer GCB/WAX SPE cartridge has equivalent performance as using dispersive GCB and is fit-for-purpose.

Analysis of a Certified Reference Material for Soil

Accuracy of analysis is important for quantitating PFAS in customer samples. A custom certified reference material (CRM) from Waters ERA was processed to demonstrate workflow accuracy. The reference material analyzed contained all 40 EPA 1633 PFAS compounds in a representative soil matrix to evaluate method performance in a known sample free from PFAS. Figure 5 shows the average quantitative results for three replicate extraction and analyses of the soil CRM. The dotted and dashed red lines indicate ±20% of the designated concentration of the CRM (solid blue line) and the solid gray line represents the average experimental quantitated value determined during sample analysis. All 40 target PFAS in EPA 1633 were quantified within ±20% of the concentration range with a mean trueness of 97% and trueness range of 85–120%. This demonstrates confidence in accuracy of the sample preparation, analysis, and data processing workflow using Waters solutions.

Sample preparation and analysis was performed for soil and fish tissue samples using EPA 1633 procedures. Oasis GCB/WAX bilayer SPE cartridges containing both WAX and GCB were utilized for the sample extraction and clean up in place of performing the extraction and clean up in two separate steps with dispersive GCB. This cartridge provides a better user experience and reduces time spent in sample preparation. All recoveries were within the acceptance criteria ranges with the mean EIS recovery of 81% and 85% for soil and fish tissue respectively. Mean RSDs were 2.8% and 9.2% for soil and tissue, respectively. This demonstrates the equivalence of the bilayer dual-phase SPE cartridge as a suitable single step replacement for the multi-step extraction and clean up presented in EPA 1633. Additionally, a Waters ERA soil reference material processed and analyzed using the same method was easily within the certified reference value range, giving high confidence in method accuracy. The data presented demonstrates that the Oasis GCB/WAX for PFAS SPE cartridge in combination with the LC-MS/MS system easily fulfills all requirements for analysis of solids and tissues for EPA 1633.

1. US Environmental Protection Agency. Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS. January 2024.
   
2. US Environmental Protection Agency. Clean Water Act Analytical Methods: CWA Analytical Methods for Per- and Polyfluorinated Alkyl Substances (PFAS). https://www.epa.gov/cwa-methods/cwa-analytical-methods-and-polyfluorinated-alkyl-substances-pfas#documents Accessed 31 Jan, 2024.
3. K Organtini, K Rosnack, P Hancock. Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Accordance with EPA 1633 Part 1: Establishing and Assessing the Method. Waters Application Note 720008117. 2023