MSACL 2025 Abstract

Sample Preparation for the Determination of an Extended Panel of Per- and Polyfluoroalkyl Substances (PFAS) from Human Serum Using UHPLC-MS/MS

Adam Senior (1), Kyle Bevan (1), Steve Plant (1), Helen Lodder (1), Lee Williams (1), Geoff Davies (1), Alan Edgington (1), Charlotte Hayes (1), Zainab Khan (1), Russell Parry (1), Lucy Richards (1), Claire Desbrow (1), Dan Menasco (1).
(1) Biotage GB Limited, Distribution Way, Dyffryn Business Park, Hengoed, CF82 7TS, UK.

Lee Williams (Presenter) Biotage GB Limited

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION
Per and polyfluoroalkyl substances (PFAS) are of concern because of their high persistence, bioaccumulation, and slow elimination with potential impacts on human and environmental health. Recently, human exposure has been linked with changes in metabolism, increased cholesterol, and high incidence of some forms of cancer. Additionally, PFAS pose particular challenges in the analytical laboratory due to their ubiquitous nature.

OBJECTIVES
This poster presents a novel method for the simultaneous determination of long and short chain PFAS in human serum at clinically relevant levels. ISOLUTE® PLD+ for PFAS, due to its optimised sorbent chemistry and functionality, allows for a simple workflow utilising solvent crash and filtration. With subtle modification to allow suitable extraction of challenging longer chain PFAS targets we have developed a robust, high sensitivity method for the clean-up of 36 PFAS compounds from human serum.

METHODS
A panel comprising 36 PFAS was spiked and extracted from human serum. Solvent crash/filtration extraction incorporating a 1:7 or 1:4 matrix/solvent ratio, utilizing ISOLUTE® PLD+ for PFAS in 96-well format was investigated. Processing was optimized using a Biotage® PRESSURE+96 positive pressure manifold. A modified procedure utilizing a 2 step sequential elution protocol was developed to optimize the extraction of longer chain 13C-18C PFAS. Extraction efficiency was compared using a range of solvents and blends of varying polarity. Final methods were developed accommodating various workflows, depending on exact application needs. Optimized methods were selected for maximum recovery and repeatability, with minimal matrix factors. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC modified with a PFAS-free flow path and a pre-injector PFAS delay column, coupled to an AB Sciex 5500 triple quadrupole MS system operating in negative ion mode.

RESULTS
A previously developed PFAS extraction protocol was used as the starting point for method development. 100 µL of serum was precipitated with acetonitrile (in a 1:7 ratio) and extracted using ISOLUTE® PLD+ for PFAS, followed by 1:1 dilution with 20 mM ammonium acetate for injection. Recoveries of carbon chains up to PFDoA (C12) were 78-90% with corresponding RSDs below 10%; however, PFAS > 12C demonstrated minimal recoveries. Matrix factors were typically 1.0-1.5 relative to dilute standards.

Extraction method modification to incorporate additional solvent volumes and/or aliquots was investigated. Addition of a second acetonitrile extraction aliquot increased recoveries of PFAS > 14C to between 5-16%. Evaluation of other solvent combinations demonstrated recovery between 4-80%, while PFODA (18C) remained unrecovered. Using a non-polar solvent as a second extraction aliquot increased recovery of PFAS >14C to between 2-82%, with PFODA recovery between 2-51%. MTBE as a second solvent provided the greatest potential for further optimization. MTBE was therefore modified with increasing proportions of methanol as a polar solvent (5-40%). These modifications demonstrated recoveries between 34-90% for longer chain PFAS, the optimum proportion was determined to be 90:10 MTBE/MeOH (% w/v).

Reinvestigation of evaporation and reconstitution conditions further enhanced sample repeatability and improved assay performance. Some longer chain PFAS RSD were approximately 15% when evaporating standards from pure solvent. Introduction of a keeper solvent to prevent full evaporation reduced RSD to below <5%. DMF proved to be the optimum keeper solvent for this assay. Workflow comparison between injecting fractions separately, combining and/or evaporation of one fraction and reconstitution with the other was evaluated.

Using the optimized protocol to extract 100 µL serum with 400 µL acetonitrile followed by 90:10 MTBE/MeOH demonstrated analyte recoveries typically 70 90% with RSD <5%. Excellent matrix depletion with respect to phospholipids, lyso-phospholipids and protein removal was also demonstrated. Full results, discussion, and conclusions will be presented in the final poster.

CONCLUSION
This poster demonstrates enhanced extraction efficiency of long chain serum PFAS in a single LC-MS acquisition, through optimized solvent selection using a multifunctional sorbent. Methods accommodate various workflows depending on exact application needs: dual acquisition for separation performance, evaporation/reconstitution for sensitivity. ISOLUTE® PLD+ for PFAS demonstrates enhanced cleanliness compared to dilute-and-shoot methodology leading to more robust methods.