Versatile TOP Assay Surrogates for Evaluating Oxidation Efficiency in Complex Environmental Samples

Accurate assessment of total per- and polyfluoroalkyl substance (PFAS) contamination remains limited because conventional targeted analyses do not capture unknown precursor compounds. The total oxidizable precursor (TOP) assay has been widely used to address this gap; however, previous studies have shown that matrix effects can cause incomplete oxidation of precursors, which can be detected with a stable-isotope labeled oxidation surrogate. Yet, current surrogates are incompatible with EPA Method 1633A. Here, we introduce a new isotopically labeled surrogate system that integrates isotopically labeled surrogates - ¹³C₃-PFHpSA (oxidation surrogate), ¹³C₃-PFHpA (transformation surrogate), and ¹³C₃-PFNA, a stable extraction surrogate that enables end-to-end tracking of method recovery across the TOP assay workflow and designed to be fully compatible with EPA Method 1633A without analytical interference. Across multiple laboratories and selected matrices, including aqueous film-forming foam (AFFF) and landfill leachate, we observed that disappearance of the oxidation surrogate does not always correspond to quantitative formation of terminal products, consistent with previously reported incidents of incomplete oxidation in complex matrices. Transformation surrogate recovery provides an additional indicator evaluating oxidation performance and potential matrix effects. These results suggest that incorporating surrogate-based quality controls can improve the performance of the TOP assay interpretation, particularly in complex environmental matrices.