Establishing Orbitrap-Based Methods for Multi-Element Compound-Specific Isotope Analysis of Per- and Polyfluoroalkyl Substances

Per- and polyfluoroalkyl substances (PFAS) are globally distributed environmental contaminants whose persistence and toxicity necessitate advanced source identification strategies. Stable isotope analysis serves as a powerful forensic tool, providing intrinsic signatures to trace contaminant origins and transformation pathways. However, multi-element isotope characterization of PFAS remains underdeveloped, with most studies limited to carbon. Here, we present a robust method for multi-element (δ 34 S, δ 15 N, and δ 18 O) compound-specific isotope analysis of PFAS using Orbitrap mass spectrometry (MS). Using perfluorobutanesulfonamide (FBSA) as a model compound, we evaluated the isotopic characteristics of its NSO 2 – fragment generated via higher-energy collisional dissociation (HCD) and in-source fragmentation. Orbitrap δ 34 S measurements across diverse FBSA samples showed high precision and good alignment with elemental analyzer–isotope ratio mass spectrometry (EA–IRMS), effectively resolving isotopic variations among samples (ranging from −4.3‰ to +4.4‰). Simultaneous δ 15 N measurements and optimized δ 18 O measurements exhibited good agreement with EA–IRMS values, demonstrating robust multi-element capability. Across 3 orders of magnitude in concentrations of FBSA (0.01 to 10 μM), Orbitrap δ 34 S values measured using HCD remained stable (2 SD = 0.96‰), whereas δ 34 S measured using in-source fragmentation at 0.01 μM deviated from the mean δ 34 S values of higher concentrations by 2.9‰. In binary mixtures with perfluorohexanesulfonamide (FHxSA), HCD-based analysis of FBSA yielded consistent isotope values, with deviations observed only under equimolar, high PFAS loading conditions (7 μM each of FBSA and FHxSA). These results demonstrate that the developed framework provides a high-resolution platform for PFAS isotope analysis, enabling new opportunities in environmental forensics.