Determination of Trace Perfluoroalkyl Acids (PFAAs) in Soil by Chelating Resin-Assisted Extraction and High-Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS)

Abstract Perfluoroalkyl acids (PFAAs) are widely distributed persistent organic pollutants and their monitoring in environmental media has received extensive attention. The soil matrix is complex, and efficient and simple extraction of PFAAs remains a challenge for analysts. In this work, a simple and rapid method has been developed for the determination of 21 PFAAs in soil by chelating resin-assisted extraction combined with high-performance liquid chromatography - mass spectrometry (HPLC-MS). Treatment with chelating resins effectively released PFAAs bound to metal ions, thereby greatly improving the extraction efficiency of PFAAs in soil. The complete extraction procedure only required 30 min. Under the optimized conditions, the quantification limits of the 21 PFAAs were from 0.03 to 0.49 ng/mL. Single PFAAs were detected in soil samples with concentrations ranging from 0.10 to 2.60 ng/g and recoveries between 70 and 126%.


Introduction
Per-and polyfluoroalkyl substances (PFASs) contain hydrophobic tails and hydrophilic functional groups.Perfluorinated alkyl acids (PFAAs) are the most abundant subclass of PFASs, including a hydrophobic polyfluoroalkyl tail and a hydrophilic carboxyl or sulfonate group (Li et al. 2022).Due to their stability and high surface activity (Abafe et al. 2021), PFASs have found a wide range of applications, including firefighting foams, paper production, food packaging, and leather (Lal et al. 2020;Liu et al. 2012;Zhou et al. 2019).They enter the soil directly from chemical plants, sewage treatment plants, landfills, or indirectly from the atmosphere.Field irrigation of contaminated surface water or groundwater is another important source of PFAAs in soil (Ghisi et al. 2019).
The PFAAs in soil may also enter the atmosphere and water through volatilization or diffusion, transfer from soil to plants, and enter organisms through the food chain.As a result, PFASs have been detected in water, air, soil and biological samples (Kim et al. 2014;Sun et al. 2011).As highly persistent and potentially hazardous substances (POPs), PFAAs accumulate in the food chain and cause a variety of health hazards.The determination of PFAAs in soil is important for understanding their migration and risk assessment.
Currently available methods for per-or polyfluoroalkyl substances (PFASs) include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography-mass spectrometry (HPLC-MS).GC and GC-MS are used to determine volatile PFASs, such as fluoromodified alcohols.The determination of nonvolatile PFASs by GC or GC-MS requires derivatization, which involves cumbersome procedures and toxic reagents.Alzaga's group developed a method for the determination of poly-fluoroalkyl carboxylic acids (PFCAs) by esterification with methyl iodide (Alzaga et al. 2005).However, most current methods for the determination of PFAAs are carried out by HPLC-MS using the multiple reaction ion monitoring mode (MRM) (Abafe et al. 2021;Deluca et al. 2021).
Due to the complex matrix of PFAAs in soil, efficient extraction must be performed before HPLC-MS analysis.These methods include Soxhlet extraction, pressurized solvent extraction, and ultrasound-assisted extraction (Mart� ınez-Moral and Tena 2012; Zabaleta et al. 2017).Some investigators believe that soil absorbs pollutants through the distribution of soil organic matter (SOM), and the absorption capacity of soil to PFASs increases with pH (Wang et al. 2022).However, it has also been reported that soil proteins better adsorb PFASs than SOM (Li et al. 2019).Table 1 summarizes efforts to improve the extraction efficiency of PFASs in soil and sediments.Common extractants are methanol, acetonitrile and methyl tert-butyl ether, and the addition of an acid or base may improve the extraction efficiency.However, overnight processing or solidphase extraction is time-consuming and inefficient.Therefore, it is necessary to develop a more efficient extraction method for PFAAs in soil.
High valence cations, such as Ca 2þ , Al 3þ, and Fe 3þ , may compensate for the negative charge from SOM or clay minerals.These cations act as metal centers that are bonded with the PFCAs (Wang et al. 2022).Therefore, the elimination of these cations may help to improve the extraction efficiency of PFCAs from soil.In our previous work, addition of ion exchange resins was found to effectively remove high valent cations from solid samples, resulting in a significant increase in the extraction efficiency of anionic phytic acid (Yu et al. 2021).
This work aimed to develop an efficient and rapid method for the determination of PFAAs in soil by chelating resin-assisted extraction.

Instrumental conditions
A centrifuge (Xiamen Lester Scientific Instruments) was used to separate soil and solution.A scanning electron microscope (SEM, Zeiss Sigma 500, Germany) and energy dispersion spectrometer (EDS, Edax, USA) were used to characterize the morphology and elemental composition of the resin.Before analysis, the microsample was placed flat on a conductive gel and coated with ion sputtering.The scanning electron microscope conditions included a test voltage of 3 kV.The test voltage for energy spectral analysis was 10 kV.The chromatographic analysis was performed using an Agilent 1,290 liquid chromatograph.Separation was performed on an XDB-C18 column (250 mm � 4.6 mm) at 30 � C, flow rate of 0.5 mL/min, and injection volume of 5 lL.Detection was carried out using the Agilent 6,495 series triple quadrupole mass spectrometer with electrospray ionization (ESI) in the negative mode using a 3,000 V capillary voltage and 20 psi nebulizing gas (N 2 ) pressure.The flow rate of the drying gas (N 2 ) was 14 L/min, its temperature was 200 � C, and the vaporization temperature was 250 � C. Multiple reaction ion monitoring (MRM) was used to scan ions.

Sample preparation
Soil samples were collected in uncontaminated polypropylene tubes and described in Supplementary Table 1.The samples were air-dried, ground, and sieved to less than 0.9 mm.0.6 g of soil powder and 0.2 g of macroporous styrene chelating resin were placed in a polypropylene tube (4 mL) and 2 mL of 70% aqueous methanol were added.The mixture was magnetically stirred for 30 min and centrifuged at 3,000 rpm for 10 min.The supernatant was passed through a 0.45 mm nylon membrane (Decima Technologies) followed by HPLC-MS analysis.
Due to their chemical structure, all PFAAs were detected as deprotonated molecules [M-H] -by HPLC-MS in the negative mode.Upon collisional activation, [M-H] -of PFCAs easily underwent CO 2 elimination for MS/MS.For example, fragmentation of [PFPeA-H] -(m/z 262.7) produced the characteristic fragment at m/z 218.8 (Figure 2a).However, it was difficult for PFSAs to undergo MS/MS fragmentation.Cleavage of [PFBS-H] -(m/z 298.7) at the collisional energy of 40 eV led to the formation of a radical anion of SO 3 -.(m/z 79.8) by losing a perfluorinated alkyl radical (Figure 2b).Based upon these results, different MRM modes were used in the MS/MS of PFCAs and PFSAs as summarized in Table 2.The fragmentation of the remaining PFAAs is shown in Supplementary Figures 1 and 2.
Using these conditions, a series of mixed standard solutions of PFCAs and PFSAs were analyzed by HPLC-MS/MS and calibration curves were constructed as summarized in Supplementary Table 2. Good linearity was achieved for both PFCAs and PFSAs with correlation coefficients (R 2 ) between 0.9,992 and 0.9,999.The limits of detection (LODs) based upon a signal-to-noise ratio of three were from 0.01 to 0.15 ng/mL.The limits of quantification based upon a signal-to-noise ratio of ten were between 0.03 and 0.49 ng/mL.Reproducibility studies were carried out by five-fold replicate determination of PFCAs and PFSAs at 0.5 ng/mL.The intra-day reproducibility (RSD) was from 1.62% and 4.88% and the inter-day reproducibility (RSD) between 1.35% and 5.54%.

Chelating resin-assisted extraction
High valent cations have been reported to inhibit the extraction of PFCAs in SOM or clay minerals (Wang et al. 2022), and thus it was essential to remove these cations to improve the extraction efficiency.Herein, D401 chelating resin was selected to remove the metal ions, and the extraction efficiency was compared with the direct extraction of  PFAAs from soil.The results show that treatment of chelating resin enhanced the extraction efficiency.In addition, several soil samples were treated with different dosages of cheating resin and the extraction efficiency evaluated by HPLC-MS.As shown in Figure 3, when the resin dosage increased from 0.0 g to 0.2 g, the PFAA content increased significantly, while the extraction results remained almost unchanged from 0.2 g to 0.4 g.
The treatment with the chelating resin increased the extraction efficiency of PFAAs by removing highly charged cations.To further investigate the mechanism of the removal of polyvalent cations, the original and the treated resins were characterized by SEM and EDS. Figure 4a shows the surface of the resin was smooth and with pores.Elemental peaks, corresponding to C, N, O and Na, are in the EDS spectrum of the resin (Figure 4c).However, particles are observed on the surface of the treated resin (Figure 4b).EDS analysis of these particles shows the presence of Fe, Ca and Mg but no F on the surface of the treated resin (Figure 4d).The results demonstrate that treatment with cationic chelating resin selectively removed multivalent cations from the soil.

Optimization of extraction conditions
Effective and selective extraction during pretreatment is essential to achieve the accurate quantification of PFAAs in soil.Several parameters were optimized, including the extractant system and stirring time.The optimization of the resin dosage is shown in Figure 3.
The extraction agent plays a key role in the effective isolation of PFAAs from soil.Herein, various methanol-water mixtures were selected to extract PFAAs.Supplementary Figure 3 shows the PFAAs content increased with the methanol composition from 0 to 50% and remained nearly constant up to 80%.70% methanol was selected to be the extraction agent in subsequent measurements.The stirring time also affects the extraction efficiency of PFAAs from soil.Supplementary Figure 4 shows the PFAAs content increased with the stirring time from 0 to 30 min and remained constant up to 2 h.Hence, the stirring time was selected to be 30 min in subsequent experiments.

Determination of PFAAs in soil
The developed procedure was employed for the determination of PFAAs in 20 soil samples.Example HPLC-MS/MS spectra are shown in Supplementary Figure 5. Table 3 shows that at least one PFAA was detected in 19 of the samples, including 10 PFCAs (C 4 -C 10 , C 12 -C 13 , C 18 ) and 4 PFSAs (C 4 -C 6 , C 10 ), indicating the prevalence of PFAAs contamination.The total concentration of PFAAs in individual soil ranged from not detected to 9.35 ng/g with PFOA having the highest content (2.60 ng/g).Short-chain PFCAs were readily detected in soil and the common substances included PFHpA, PFDoA, PFBS, and PFHxA.These results indicate that the short-chain PFCAs were alternatives to traditional PFOAs (Ma et al. 2022), following the listing of the latter on the Stockholm Convention's list of persistent organic pollutants (Xu et al. 2021).
The recoveries were from 70% to 126% as shown in Supplementary Table 3, which was similar to the literature, indicating the reliability of this developed approach.The chelating resin-assisted extraction significantly reduced the pretreatment time to 30 min, providing a simple and efficient method for PFAAs in soil.The analyte abbreviations are defined in Table 2.

Conclusion
A simple and rapid method has been developed for the determination of PFAAs in soil with HPLC-MS determination.The deprotonated PFCAs readily undergo CO 2 elimination in MS/MS, while it was difficult for deprotonated PFSAs to undergo fragmentation.Thus, decarboxylated ions of PFCAs and deprotonated ions of PFSAs were selected to be the quantitative ions for MRM analysis.The limits of quantification of the PFAAs ranged from 0.10 to 1.63 ng/g and the recoveries between 70 and 126%.
Treatment of chelating resin effectively replaced PFAAs that were bound to metal ions in the soil, thereby improving the extraction efficiency of the PFAAs.The preparation time was only 30 min, compared to 24 h by conventional methods.Using the optimized conditions, the PFAAs were determined in soil: 19 of 20 samples contained the analytes, with PFPeA, PFHxA, PFHpA, PFDoA, and PFBS the primary components.This work may serve as a reference for subsequent work involving the pretreatment of soil.

Figure 1 .
Figure 1.Chromatographic separation of 100 ng/mL PFAAs by HPLC-MS.The analyte abbreviations are defined in Table2.

Figure 3 .
Figure 3. Influence of the resin dosage upon the extraction efficiency of 1 ng/mL PFAAs.

Figure 4 .
Figure 4. Scanning electron microscopic images of the (a) original resin and (b) resin treated with soil.Energy dispersion spectra (EDS) of (c) original resin and (d) resin treated with soil.

Table 1 .
Comparison of the analytical figures of merit of the developed protocol with the literature for the determination of PFASs in soil and sediments.

Table 2 .
Dynamic multiple reaction monitoring parameters for the HPLC-MS/MS determination of PFAAs.