Advanced LC–MS Analysis for PFAS Analysis in Eggs

Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals that have been widely used since the 1940s due to their unique properties, including resistance to heat, water, and oil. These characteristics have led to their extensive use in various commercial applications such as nonstick cookware, food packaging, firefighting foams, and textiles. However, PFAS are persistent in the environment and can accumulate in living organisms, leading to potential health risks. Consequently, there is a growing need for stringent monitoring of PFAS in food products to ensure public safety.

The European Commission’s regulation (2023/915) (1) describes the maximum levels in fish, meat, and egg products for four PFAS components. The maximum tolerated levels are for PFOS, PFOA, PFNA, and PFHxS, at levels of 1.0, 0.30, 0.70, and 0.30 μg/kg, respectively. Additionally, the sum of the four components has a maximumvlevel of 1.7 μg/kg in eggs. Other than this regulation, there is also an EU CommissionvRecommendation (2022/1431) in place on the monitoring of PFAS substances in foodv(2). This recommendation mentions that member states should monitor, if possible, the presence of compounds that are similar to PFOS, PFOA, PFNA, and PFHxS, and suggests 18 different components in this regard.

Analysis of eggs can be challenging due to the presence of matrix interferences such as cholesterol, lipids, bile acids, and proteins. The technique used in this workflow includes efficient sample cleanup using QuEChERS in combination with Agilent Bond Elut Carbon S solid phase extraction cartridges followed by LC–MS/MS analysis.

Experimental

Sample Preparation

Homogenization and Extraction

Egg samples are homogenized to ensure uniformity. A Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction method is employed to isolate PFAS from the egg matrix. This involves adding acetonitrile and formic acid to the homogenized samples, adding QuEChERS salts, followed by shaking and centrifugation to separate the supernatant. Additionally, a QuEChERS dispersive SPE kit was used.

Solid-Phase Extraction (SPE)

The supernatant undergoes further cleanup using carbon-based SPE cartridges. This step is crucial for removing matrix interferences such as cholesterol, lipids, and proteins that can affect the accuracy of the analysis.

LC–MS Analysis

Instrumentation

The analysis is conducted using a high-performance liquid chromatography (HPLC) system coupled with a triple quadrupole mass spectrometer (MS). This setup allows for the separation, detection, and quantitation of PFAS compounds with high sensitivity and specificity.

dMRM Settings

Dynamic multiple reaction monitoring (dMRM) settings are optimized to ensure precise detection and quantitation of the target PFAS compounds. This involves selecting specific precursor and product ion pairs for each compound, which enhances the selectivity and sensitivity of the method.

Chemicals and Reagents

LC–MS-grade acetonitrile and methanol were used, along with ultrapure water, ammonium acetate, formic acid, and ethylene glycol. These high-purity reagents are essential for minimizing background contamination and ensuring accurate results. PFAS standards and internal standards were obtained from a reputable supplier. These standards are critical for ensuring the accuracy and precision of the analysis.

Sample Extraction Workflow Homogenization

Egg samples are homogenized to ensure uniformity and consistency in the extraction process. QuEChERS Extraction: A QuEChERS extraction method is employed to isolate PFAS from the egg matrix. This involves adding acetonitrile and formic acid and QuEChERS salts to the homogenized samples, followed by shaking and centrifugation to separate the supernatant. After this, the supernatant is added to a dispersive SPE QuEChERS kit.

Solid-Phase Extraction (SPE)

The supernatant undergoes further cleanup using carbon-based SPE cartridges. The combination of organic solvent, QuEChers salts, dSPE and carbon-based SPE is crucial for removing matrix interferences such as cholesterol, lipids, and proteins that can affect the accuracy of the analysis.

Preparation of Calibration Standards and QC Samples

Calibration standards, matrix blanks, and quality control (QC) samples are prepared to ensure the accuracy and precision of the analysis. These standards and samples undergo the same extraction and cleanup procedures as the egg samples (Table 1). Table 1 shows the calibration curve ranges per component, the concentration of the QC samples as well as the used internal standards and retention times.

LC–MS Instrumentation

Quantitative analysis was performed by LC–MS/MS system using the 1290 Infinity II LC system coupled to a 6475A triple quadrupole LC–MS (Agilent). The UHPLC system includes a kit to reduce PFAS background. These components ensure efficient separation of PFAS compounds and additionally reduce PFAS background.

Method Validation Parameters

Calibration Performance

Calibration curves for all analytes showed high linearity, with R² values greater than 0.99. This indicates that the method provides accurate and consistent quantitation across a wide range of concentrations. Six-point calibration curves were used, with concentration ranges tailored for each PFAS component. This ensures that the method is sensitive enough to detect low levels of PFAS while maintaining accuracy at higher concentrations.

Recovery and Precision

Recovery rates for all PFAS components ranged from 93.5% to 109.0%, within the acceptable limit of 70% to 120%. This indicates that the extraction and cleanup procedures are effective in isolating PFAS from the egg matrix. Precision, measured as relative standard deviation (RSD), was below 10.3% for most compounds, except for PFOSA, which had an RSD of 17.5% due to the lack of a corresponding stable label internal standard. This demonstrates the method’s reproducibility and reliability.

Matrix Effect

Minimal matrix effects were observed, with peak responses for extract spike samples comparable to system suitability samples (PFAS components in solvent). This indicates that the method is robust and can accurately quantify PFAS in the presence of complex matrix components.

Method Detection Limits

The lowest calibration standard was used to determine method detection limits, with deviations from back-calculated concentrations within ±15.2%, well below the acceptable limit of ±20%. This demonstrates the method’s sensitivity and ability to detect low levels of PFAS.

Results and Discussion

The method was validated according to SANTE 11312/2021 guidance ensuring its reliability for routine analysis (3). All analytes had consistent retention times with RSDs over the entire run of less than 0.05%. All analytes also had excellent calibration curve R² values of greater than 0.99 for a six-point curve using linear fit with no weighting, except for PFHpS and PFPeS, where a weighting of 1/X was applied. The MRM chromatogram shown in Figure 1 demonstrates good separation and detection of the target PFAS. Table 1 shows the calibration curve ranges per component, the concentration of the QC samples as well as the used internal standards and retention times.

Table 2 shows excellent recovery rates, ranging from 93.5% to 109.0%, well within the acceptable limit of 70 to 120%, indicating that the extraction and cleanup procedures were effective in isolating PFAS from the egg matrix. Precision, measured as relative standard deviation (RSD), was below 10.3% for most compounds, except for PFOSA, which had an RSD of 17.5% due to the lack of a corresponding stable label internal standard. Calibration curves showed high linearity, with R² values greater than 0.99, and the method detection limits were well within SANTE acceptable value of ≤ 20%.

Figure 2(a) shows chromatograms of PFOS, PFOA, PFNA, and PFHxS (the four PFAS components in the EU regulation) in addition to two other groups, PFNS and PFHxA, at the lowest calibration level. To demonstrate the selectivity of the method, the chromatograms of the method blank are shown in Figure 2(b): A small amount of PFOA is visible in the method blank, but for all other PFAS components, the method blank did not show any peaks. The samples used for the SANTE validation were crosschecked on two different LC–MS/MS systems in two different laboratories.

The combination of QuEChERS extraction and SPE cleanup effectively addresses the challenges of analyzing PFAS in complex matrices like eggs. The QuEChERS method is advantageous due to its simplicity, cost-effectiveness, and ability to handle a wide range of sample types. The use of carbon-based SPE cartridges further enhances the cleanup process by efficiently removing matrix interferences that can compromise the accuracy of the analysis.

The LC–MS system provides the sensitivity and specificity required for regulatory compliance. The HPLC system ensures efficient separation of PFAS compounds, while the triple quadrupole mass spectrometer allows for precise detection and quantitation. The optimized MRM settings enhance the selectivity and sensitivity of the method, enabling the accurate quantitation of PFAS at low concentrations.

This method is now routinely used for monitoring PFAS in eggs, contributing to food safety and public health. The ability to accurately quantify PFAS in food products is essential for ensuring compliance with regulatory standards and protecting consumers from potential health risks associated with PFAS exposure.

Conclusion

The validated method offers a reliable solution for quantifying PFAS in eggs, meeting the stringent requirements of the European Commission’s regulations. The use of advanced LC–MS technology ensures accurate and reproducible results, supporting ongoing efforts to monitor and control PFAS contamination in food. This method can be adapted for other food matrices and environmental samples, providing a comprehensive approach to PFAS monitoring. The ongoing development of regulations worldwide underscores the importance of such robust analytical methods.

References

(1) Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels of certain contaminants in food repealing Regulation (EC) No 1881/2006.

(2) Commission Recommendation (EU) 2022/1431 of 24 August 2022 on the monitoring of perfluoroalkyl substances in food.

(3) Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed; SANTE 11312/2021.

About the Authors

Carola Damen obtained her MSc in pharmacy at the University of Groningen, after which she completed her PharmD degree. During her MSc and PhD studies she specialized fully in the field of analytical chemistry and in LC–MS. Currently she works as a product specialist LC–MS at Agilent Technologies and has a special point of interest the analysis of PFAS. Correspondence: carola.damen@agilent.com

Peter Kornas graduated as a Food Chemist at the Goethe University Frankfurt. He has over a decade of experience in the development and validation of analytical methods for pesticide, mycotoxin, PFAS, and contaminant analysis using LC–MS. He currently works at Agilent Technologies as an LC–MS Application Engineer.

René van der Molen obtained his Bsc degree at Hogeschool Van Hall Larenstein (Leeuwarden) in Forensic Sciences where he specialized in analytical chemistry. Currently, he works as an analytical chemist and LC–MS operator at Ducares B.V., Trading as Triskelion. He is specialized in the analysis of small molecules in complex veterinary matrices.