Automated SPE Extraction and UPLCMS/ MS Analysis PFAS in Milk

Significance of the Topic

The analysis of per‐ and polyfluoroalkyl substances (PFAS) in milk is critical due to their persistence, bioaccumulation and potential adverse health effects. Milk represents a key exposure pathway, especially for infants and young children. Regulatory bodies in the EU have established stringent indicative levels for PFAS in foodstuffs under Recommendation 2022/1431. Implementing reliable, high-throughput methods ensures consumer safety and regulatory compliance.

Objectives and Study Overview

This work aimed to develop and validate a fully automated workflow for solid‐phase extraction (SPE) and UPLC‐MS/MS analysis of PFAS in milk. The specific goals were:

• Automate calibration standard preparation and SPE sample cleanup using the Andrew+ pipetting Robot and Extraction+ device.
• Integrate Oasis GCB/WAX cartridges for efficient PFAS extraction.
• Couple the workflow with ACQUITY Premier UPLC and Xevo TQ Absolute MS for sensitive quantification.
• Validate performance to meet EU Commission Recommendation 2022/1431 and EURL POP guidance.

Methodology

Milk samples (2.5 g) were spiked with isotopically labeled internal standards and acidified. Biphasic extraction with formic acid and acetonitrile was followed by centrifugation and combination of supernatants. Automated SPE used dual‐phase Oasis GCB/WAX cartridges primed and washed under programmed vacuum profiles to optimize flow and minimize blockages. Elution with methanolic ammonium hydroxide, evaporation and reconstitution completed sample prep. Calibration standards (0.00025–1 µg/kg equivalent) were created by automated serial dilution.

Used Instrumentation

• Andrew+ Pipetting Robot with Extraction+ connected device
• Oasis GCB/WAX SPE cartridges
• ACQUITY Premier UPLC System with PFAS Analysis Kit
• Xevo TQ Absolute Triple Quadrupole Mass Spectrometer
• waters_connect informatics platform

Main Results and Discussion

Automating 337 pipetting and extraction steps for 12 samples saved ~3.5 hours of analyst time and reduced inter‐operator variability. Calibration curves for PFOS, PFOA, PFNA and PFHxS showed R2 > 0.99 with residuals within ±20%. Limits of quantification were validated at 0.005 µg/kg in milk, exceeding EU requirements. Recoveries ranged 98–118% for four mandatory PFAS; overall mean recovery across 28 PFAS was 102 ± 18%. Process blanks confirmed low background contamination, with methanol rinse steps and drip‐through design minimizing PFAS carryover. A within‐laboratory validation across five milk types and two operators demonstrated robust precision (≤ 20%) and trueness (± 20%), except in heavily flavored milk where matrix effects elevated PFOS recoveries.

Benefits and Practical Applications

• High automation level reduces manual errors and increases throughput.
• Enhanced sensitivity meets challenging LOQs for milk matrices.
• Standardized protocols improve inter‐laboratory reproducibility.
• Modular workflow adaptable to other food matrices (fruits, vegetables, baby food).

Future Trends and Applications

Emerging trends include expanding automated SPE‐UPLC‐MS/MS workflows to broader PFAS panels and complex matrices. Integration with advanced robotics and AI‐driven method optimization will further enhance throughput. Development of isotopically labeled standards for all PFAS analytes and advanced sample cleanup techniques will address matrix effects in heavily processed foods.

Conclusion

The automated SPE and UPLC‐MS/MS workflow delivers reliable quantification of PFAS in milk at sub‐µg/kg levels, meeting EU directives while saving significant analyst time. The approach enhances reproducibility, minimizes contamination risks and can be extended to other food safety applications.

References

1. EU Commission Recommendation 2022/1431 on PFAS monitoring in food.
2. EURL POPs Guidance Document on PFAS in Food and Feed (May 2022).
3. Rawn et al., Sci Total Environ. 831:154888 (2022).
4. Dreolin et al., Waters Application Note 720008219 (2024).
5. Danaceau & Trudeau, Waters Application Note 720007712 (2022).