Preserved and Accessible: Quantifying PFAS in Formalin-Fixed and Paraffin-Embedded Tissues for Retrospective Exposure...

Background: Formalin-fixed (FFT) and formalin-fixed paraffin-embedded (FFPET) tissues represent an immense and underutilized resource for reconstructing historical chemical exposures. For persistent contaminants such as per- and polyfluoroalkyl substances (PFAS), these archived specimens could enable retrospective biomonitoring and tissue-level dosimetry, linking exposure history to disease outcomes. However, fixation and embedding may alter analyte stability and recovery, creating uncertainty in quantification. Methods: We have developed and tested a liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method to quantify six commonly found environmentally relevant PFAS (PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA) in mouse liver, kidney, ileum, and brain. Tissues from PFAS-exposed mice were divided into matched sets of flash-frozen tissues (FT), formalin-fixed (FFT), and formalin-fixed paraffin-embedded (FFPET) samples. PFAS recovery and loss were tracked through fixation, storage, and deparaffinization steps. Results: Across matched flash-frozen tissues, the mean sum of PFAS ranged from 279.2 ng/g in the brain to 22,628.4 ng/g in the liver. All PFAS were quantifiable in each matrix. Formalin fixation largely preserved PFAS, with recoveries of 76.7-83.8% relative to fresh tissue, while 12.6-19.9% of PFAS were detected in the formalin solution. Paraffin embedding resulted in further losses, yielding 57.4-75.1% recovery, with 6.1-15.9% loss during xylene and ethanol processing. Longer-chain PFAS (PFNA-PFUnA) exhibited greater retention than shorter-chain species (PFHxS), and tissues with higher PFAS burdens, such as liver, showed better recovery than low-burden tissues like brain and ileum. Conclusion: This work demonstrates the feasibility of quantifying PFAS in archived tissues and provides the first systematic insight into compound- and matrix-specific recovery patterns. Further validation is needed for short and ultrashort PFAS. Human tissues with much lower PFAS loads may experience proportionally greater analytical losses, requiring higher analytical sensitivity and application of correction factors for accurate dose estimation.