Esraa Abojasser, Lee Williams, Adam Senior and Kyle Dukes

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, 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. We have optimized an automated and robust, high-sensitivity method for the clean-up of 31 PFAS compounds from various biological fluids. 

Methods: A suite comprising 31 PFAS was spiked and extracted from human serum, plasma, whole blood, and urine. Solvent crash/filtration extraction incorporating a 1:7 matrix/solvent ratio, utilizing ISOLUTE PLD+ for PFAS in 96-well format was investigated. Solvent crash performance was compared using solvent/matrix first approaches. Optimized methods were selected for maximum recovery and repeatability, with minimal matrix factors. Final protocols were used to determine analyte linearity and sensitivity. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC and a Sciex 5500 triple quadrupole MS. 

Results: Serum recoveries were 80-94%, typical RSD were <5%, with matrix factors typically1.0-1.5.. Plasma recoveries from 100 μL matrix using automation were 77-80%, with RSD <5%, matrix factors were 1.0-1.5; manual extraction recoveries were 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 1.2-1.5. Manual recoveries were 82-94%, with matrix factors 1.3-1.5, RSDs 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 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. Most analytes 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 increased variation. ISOLUTE PLD+ for PFAS demonstrates excellent matrix depletion compared to centrifugation and dilute/shoot. The optimized extraction protocol results in 99.9% depletion of phospholipid and lysophospholipids, while effecting efficient protein removal. Analytical column lifetime is improved by minimizing matrix build-up over multiple injections, maintaining analyte sensitivity over extended analytical runs.