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Introduction

Per and Poly-fluoroalkyl substances (PFAS) are synthetic compounds that are found in a wide range of industrial and consumer products. Due to the strong nature of the carbon-fluorine bond, these compounds are resistant to degradation and have been found to accumulate in fish, wildlife and multiple environmental samples (ex. water, soil…), posing a significant health risk to humans. Current sample preparation techniques for PFAS analysis are laborious and not easily automated. In this study, supercritical fluid extraction (SFE) was evaluated as an alternative sample preparation technique for the extraction of eighteen PFAS compounds from fish tissue, as a preconcentration step prior to their analysis by LC-MS/MS.

Experimental Approach

For this study, the Shimadzu Nexera UC offline SFE system (configuration shown in Figure 1) was employed. 0.5 grams of freeze-dried fish tissue was milled and mixed with 1 packet (1 gram) of Miyazaki Hydro-Protect and placed into a 5 mL extraction vessel for extraction.

Optimized extraction conditions to maximize PFAS recoveries are shown in Table 1. After extraction, the sample was dried down under nitrogen and reconstituted with 1 mL of methanol. The sample was centrifuged and the supernatant was transferred to an LC vial. 1 uL of the supernatant was injected for LC-MS/MS analysis. Table 2 shows the LC-MS/MS conditions used for the Shimadzu LCMS-8050 for this study; a representative chromatogram is included in Figure 2.

Table 1: SFE optimized method conditions

Table 2: LC-MS/MS method conditions used in Shimadzu LCMS-8050

Results and Discussion

Recovery, Linearity, Reproducibility

A set of experiments to identify the combination of CO₂’s modifier and additives that maximized the extraction efficiency of 18 PFAS was first conducted in this work. While 100% CO₂ can be effective in extracting nonpolar compounds, the addition of a cosolvent is often required in SFE to extract more polar compounds. Optimum extraction conditions were found to be 20% methanol without the need for additives. The 18 targets from this study showed recoveries over 95% with these conditions, as shown in Table 3.

Linearity of a matrix matched calibration curve, to minimize the impact from coextracted matrix components, was evaluated. Concentrations from 0.5 to 100 ng/g were spiked to a freeze-dried farm-raised trout fish tissue sample found to be free from PFAS contamination.

Linearity results are shown in Table 4 along with the determined limit of quantitation for each compound; r² for all compounds was >0.9995 except for N-MeFOSAA (r²: 0.9994). Linearity results show accurate determinations for PFAS compounds can be obtained regardless of concentration levels.

Reproducibility results for supercritical fluid extractions were determined at three PFAS concentration levels: 2 ng/g, 20 ng/g and 100 ng/g. Extractions were performed in triplicated samples. Table 5 summarizes the variability of the extraction at each of the concentrations evaluated. %RSDs at 20 and 100 ng/g were less than 12% for all compounds evaluated. At 2 ng/g, %RSD was less than 25%, except for PFTriA (27%) and N-MeFOSAA (45%). These results demonstrate the reproducibility of SFE as a sample preparation technique.

Quantitative Analysis of Fish Samples

Three fish samples with unknown PFAS concentrations were then evaluated with this method. The samples were wild caught Walleye, wild caught Large Mouth Bass, and farm raised Trout. Figure 3 shows the LC-MS/MS chromatogram of an extracted sample from each fish’s type. Table 6 shows the concentration of PFAS determined in each type of fish. The wild caught Walleye and Large Mouth Bass were found to contain the largest amounts of PFOS, PFDA, and PFUnA. No PFAS compounds were detected above the LOQ in the farm raised Trout sample.

Table 4: Linearity of PFAS compounds spiked onto fish tissue

Table 5: Reproducibility of PFAS SFE extractions (n=3)

(a) Wild caught Large Mouth Bass

(b) Wild caught Walleye

(c) Farm raised Trout

Figure 3: SFE extracted sample chromatograms from (a) Wild caught Large Mouth Bass, (b) Wild caught Walleye, and (c) Farm raised Trout

Table 6: Concentration of 18 PFAS in unknown fish samples

Conclusion

A novel supercritical fluid extraction method, using the Shimadzu Nexera UC offline SFE system, for the extraction of PFAS compounds from fish tissue was evaluated and provided excellent results for recovery, linearity, and reproducibility. The results summarized here demonstrate the suitability of SFE as a sample preparation technique for PFAS analysis.

This sample preparation technique can be automated to allow the processing of up to 48 samples per batch to help reduce manual labor in testing laboratories.

Acknowledgement

Shimadzu acknowledges Pennsylvania Department of Environmental Protection Bureau of Laboratories for providing the samples analyzed in this study.