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Abstract INTRODUCTION: Exposure to Per and polyfluoroalkyl substances (PFAS) has been linked with changes in metabolism, increased cholesterol, and high incidence of some forms of cancer. PFAS pose particular challenges in the analytical laboratory due to their ubiquitous nature. Environmentally, PFAS are of concern because of their high persistence (forever chemicals), bioaccumulation and slow elimination, and potential impacts on human and environmental health. We present automated methods to determine clinically relevant levels of PFAS in common biological matrices.
OBJECTIVES: This poster presents an automated and robust, high-sensitivity method for the clean-up of 31 PFAS compounds from various biological fluids. ISOLUTE® PLD+ for PFAS, due to its optimised sorbent chemistry and functionality, allows a simple solvent crash/filtration-based approach.
METHODS: A suite comprising 31 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. Simple solvent crash/filtration extraction methodologies incorporating a 1:7 matrix/solvent ratio, utilizing ISOLUTE® PLD+ for PFAS in 96-well format were investigated. Processing parameters were optimized for the Biotage® Vacmaster™-96 vacuum manifold and the Biotage® Extrahera™ automated sample preparation platform at 100 µL and 50 µL sample volumes. Solvent crash performance was compared using solvent/matrix first approaches. Extrahera™ automation parameters were optimised for each matrix to maximise sample/solvent crash and mixing efficiency, and included system consumables, pipetting parameters (aspirate/dispense frequency, height and speed), and extraction conditions (applied pressure and duration). The optimized Vacmaster™ and Extrahera™ methods for each matrix were selected on the basis of maximum recovery and repeatability in combination with minimal matrix factors. Final ISOLUTE® PLD+ for PFAS protocols for manual and automated platforms were used to assess method performance at 100 µL and 50 µL load volumes for all matrices. Target analyte linearity was determined from 0.1 to 100 ng mL-1 over 8 levels. Sensitivity (limit of quantitation, LOQ) was estimated where signal/noise of the lowest calibration standard was >10:1, with accuracy of 80-120% and RSD <15%. 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: Optimized extraction protocols utilizing ISOLUTE® PLD+ for PFAS sample preparation demonstrate highly consistent recoveries and high repeatability for all PFAS in our target suite. Using the Extrahera™ automated sample extraction platform and 100 µL sample volumes, serum recoveries were 80-94%, typical RSD were <5%, with matrix factors typically1.0-1.5; sample extraction using Vacmaster™ demonstrated comparable performance. Results obtained using 50 µL sample volumes were similar, automated recovery RSD were lower than demonstrated from manually extracted samples. Plasma recoveries from 100 µL matrix using automation were 77-80%, with RSD <5%, matrix factors were 1.0-1.5; manual extraction recoveries demonstrated greater variability (58-89%), with correspondingly higher RSD (< 10%), matrix factors were comparable. Automation recoveries from whole blood were 79-90%, typical RSD were <5%, matrix factors were slightly higher than from blood products (1.2-1.5), probably due to increased matrix complexity. Manual recoveries were slightly higher than automated (82-94%), as were matrix factors (1.3-1.5), RSD were comparable to automated extraction. Similar results for whole blood were demonstrated using 50 µL sample volumes, automated RSD were lower than manual RSD. Urine recoveries using our automated protocol were 70-82%, RSD were <6%, and MF 1.0-1.5; manual recoveries were comparable (75-90%) with RSD <6%, matrix factors 1.0-1.5 were typically observed. Results obtained using 50 µL urine demonstrated less consistent results with greater variability. The optimized ISOLUTE™ PLD+ for PFAS protocol was used to determine extracted linearity and calibration ranges. Method performance data were similar using 100 µL matrix volumes with Extrahera™ automation or Vacmaster™ manual extraction. Most analytes in our target suite demonstrate LOQ at 0.1 ng mL-1. All analytes demonstrate good linearity, r2 > 0.995. Most analytes demonstrate repeatability <10% (<20% at LOQ). Typical analyte accuracy was 90-110% (80-120% at LOQ). Method performance using 50 µL matrix volumes is similar to 100 µL with slightly increase variation, probably due to increased variation in liquid transfer. ISOLUTE™ PLD+ for PFAS demonstrates excellent matrix depletion compared to centrifugation and dilute/shoot. Our optimized extraction protocol results in 99.9% depletion of phospholipid and lysophospholipid, almost all protein is removed. Analytical column lifetime is improved by preventing matrix build-up over multiple injections, maintaining analyte sensitivity over extended analytical runs.
CONCLUSION: Optimized extraction protocols utilizing ISOLUTE® PLD+ for PFAS demonstrate consistent PFAS recovery and sensitivity, with low matrix factors, for 31 PFAS residues extracted from a variety of biological fluids. Use of the Biotage® Extrahera™ automated sample preparation platform demonstrates comparable data to a manual vacuum-based method, helping to improve sample throughput. ISOLUTE® PLD+ for PFAS demonstrates good accuracy and precision across the calibrated range of the assay, with LOQ at clinically relevant levels. Due to its optimised sorbent chemistry and functionality, ISOLUTE® PLD+ for PFAS demonstrates enhanced cleanliness compared to diMlute-and-shoot methodology leading to more robust methods. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
