Annotating DHA Metabolites Interfering with the Quantification of Emerging Perfluoroalkyl Ether Carboxylic Acids

Quantitative analyses of per- and polyfluoroalkyl substances (PFAS) commonly rely on low-resolution targeted tandem mass spectrometry (MS/MS) due to its high sensitivity and relatively low operational threshold. Perfluoro-3,5,7,9-butaoxadecanoic acid (PFO4DA), an important constituent of the perfluoropolyether carboxylic acid (PFECA) family, has been widely detected in biotic and abiotic matrices. Nevertheless, marked interference has been observed in the MS/MS transition for PFO4DA (377 > 85) in biological samples, particularly in aquatic organisms, resulting in substantial overestimation of concentrations, reaching up to 66 ng/g. In this study, nontargeted molecular networking strategies, combined with the spectral simulation tools SIRIUS and QCxMS, were applied to identify the source of interference, which was confirmed as an oxygenated metabolite of docosahexaenoic acid (DHA) based on fragmentation patterns matching reference standards. The study further clarified that fatty acids generated the fragment ion [C₄H₅O₂]⁻ (85.0296 Da), which overlapped with the [CF₃O]⁻ (84.9907 Da) ion of PFECA compounds under low-resolution conditions, causing signal interference. Analysis of river water and fish samples revealed that this interference led to a 24-fold overestimation of PFO4DA bioaccumulation in aquatic organisms. These findings are essential for improving the selectivity of environmental exposure assessments for PFECA compounds.