MSACL 2023 Abstract

Extraction and Quantitation of Per and Polyfluoroalkyl Substances (PFAS) in Bioanalytical Matrices Determined Using UHPLC-MS/MS

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

Adam Senior, BSc Natural Science with Chemistry (Open) (Presenter) Biotage GB Ltd

Presenter Bio: Experienced analytical scientist with extensive laboratory skills in sample preparation and compound separation for over twenty years. A technology enthusiast who is constantly seeking and advocating new approaches to current challenges.

Specialties: RP, IEX, IC and GPC liquid chromatography using fluorescence, UV, electrochemical and MS/MS detection; MS/MS method development using triple quadrupole and ion trap systems; project resource planning; report and protocol writing; specification and purchase of instrumentation.

Relevant Financial Disclosures (within past 24 months, reported on Jul 17, 2025) Salary Biotage GB ltd Conflict mitigation NOT REQUIRED.

Introduction
Per and polyfluoroalkyl substances (PFAS) comprise a large number of compounds that occur in a broad range of applications and products. PFAS are of concern because of their high persistence, bioaccumulation and slow elimination, and impacts on human and environmental health. Exposure to PFAS correlates with changes in metabolism, higher cholesterol, and increased risk of some cancers. PFAS pose particular challenges in the analytical laboratory as they are present in common consumables and hardware. We present methods to determine clinically relevant levels of PFAS in common biological matrices.

Objectives
This poster will present a consistent approach for robust high-sensitivity clean-up of PFAS including Gen X from biological matrices utilising a multifunctional sorbent bed in conjunction with a solvent crash/filtration-based procedure. Analytical column lifetime is improved by preventing matrix build-up over multiple injections, while maintaining analyte sensitivity over extended analytical runs.

Methods
A suite comprising 25 target analytes from 10 classes of PFAS was spiked and extracted from human serum, plasma, whole blood, and urine matrices. The targets varied by functionality, including carboxylic acids, sulfonic acids and telomers, sulfonamides, and ethoxy compounds. Sample extraction was investigated using polymer-based solid phase extraction and compared to sample clean-up using multifunctional sorbent strategies. Extraction solvents and crash/pre-treatment ratios to matrix volume were compared. Solvent pH modifiers, and matrix/solvent first protocols were also investigated where appropriate. Extract evaporation and reconstitution was compared to dilution and injection protocols using differing reconstitution/dilution solvents. Methods were optimized for plasma and whole blood matrices. We selected the best performing sample preparation methodology for maximum recovery and repeatability in conjunction with minimal matrix factors. Vacuum processing with a Biotage® Vacmaster™ 96 manifold was used throughout to minimize the number of potential contact surfaces for PFAS transfer. Final extraction protocols using a novel sample clean-up plate in a 96-well format were used to determine target analyte linearity and sensitivity at 100 µL and 50 µL load volumes for all matrices. 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
The target suite of 25 PFAS was extracted from serum and urine using polymer-based reversed phase and corresponding mixed-mode weak anion exchange SPE chemistries in 30 mg 96-well plate formats. Reverse-phase chemistry demonstrated recovery below 50% for some long-chain PFAS with correspondingly high RSD. Modification of load, wash, and elution steps did not demonstrate improvements in method performance. Weak anion exchange (WAX) SPE chemistry demonstrated comparable method performance to reverse-phase SPE. Short-chain PFAS recoveries were typically above 95%, with low RSD and matrix factors close to 1. No improvement to WAX recovery was demonstrated from method modifications. The same suite was extracted using a novel multifunctional sorbent bed in 96-well plate format using 100 µL serum and urine matrix volumes. We found optimum extraction was obtained using a solvent first protocol in a 1:7 ratio with unmodified acetonitrile, target analyte recoveries were consistently above 80% with most RSD below 5%. Evaporation/reconstitution of extracts demonstrated no recovery of some short-chain PFAS and RSD over 10% for nearly half the extracted suite. A dilute/inject strategy with dilute ammonium acetate demonstrated consistent recoveries above 80%. We demonstrate optimized recoveries typically above 80% across our target suite for all matrices. Matrix factors between 0.8 and 1.3 were demonstrated for most analytes. Our extraction method is highly reproducible, the majority of RSD are demonstrated below 5%. We demonstrate sub ng mL-1 LOQs for most analytes in all matrices, at clinical levels similar to those demonstrated from NIST SRM 1957. Calibration curves were linear over our extracted range (0.1 to 100 ng mL-1), coefficients of determination (r²) were above 0.99 for all targets while demonstrating excellent matrix reduction, removing >99.9 % phospholipids where present. Background levels of PFAS in pooled and individual donor matrices tested were in the order of 1 ng mL-1. This novel media demonstrates PFAS background well below reportable limits. Low PFAS residues are vital to ensure results are reproducible and free from product-derived interference.

Conclusion
We demonstrate high PFAS analyte recovery and sensitivity with low matrix factors and repeatability. This methodology demonstrates more consistent performance compared to other commonly used sample preparation techniques: greater cleanliness than dilute-and-shoot methodology leading to more robust methods, and consistent recovery compared to reverse-phase or mixed-mode SPE methodology.