Novel LC-MS/MS Method for Monitoring Perfluoroalkyl and Polyfluoroalkyl Substances in Companion Animal Blood Plasma and Serum: Environmental and Health Implications

Background: Per- and poly-fluoroalkyl substances (PFAS), known as "forever chemicals," are synthetic compounds widely used for their unique physicochemical properties. Their environmental persistence and potential link to adverse health outcomes have raised significant concerns, necessitating robust monitoring methods. While numerous studies have quantified PFAS in human biological fluids, there is a critical gap in the availability of validated analytical techniques for companion animals remain scares. Aims and Objectives: The aim of this study was to develop and validate a novel analytical method for the quantification of eleven PFAS in biological samples from companion animals, specifically canine serum and feline plasma, and to apply this method to real samples. Materials and Methods: A streamlined sample preparation protocol was developed including protein precipitation, evaporation under nitrogen at 45°C, and reconstitution. Quantification was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry in negative electrospray mode, monitoring two transitions for each compound. Results: ² ≥ 0.99), high precision (CV% < 15%), and accuracy within ±15% of nominal concentrations. Recovery ranged from 80.0% to 114.9% and process efficiency from 80.7% to 114.0%, with negligible matrix effects and no observable carry-over. Application to real samples from 23 healthy dogs and 4 healthy cats revealed PFAS concentrations ranging from 0.1 to 15.8 µg/L in dogs and from 0.1 to 2.8 µg/L in cats. PFOS was the most prevalent compound in both species, with PFHxS also prominent in cats. Conclusion: The validated UHPLC-MS/MS method provides a reliable and efficient tool for the biomonitoring of PFAS in companion animals. Its application enables large-scale assessment of environmental exposure and supports the evaluation of potential health implications for both pets and their owners.