Critical Reassessment of Anthracene Probes for Singlet Oxygen Detection in AOPs through Reference-Based LC-MS/MS Fingerprinting

Abstract Singlet oxygen (1O2) is frequently invoked as a key reactive species in water treatment advanced oxidation processes (AOPs), yet its reliable assignment has become increasingly challenging, with evidence of false-positive interpretations in conventional detection approaches. Anthracene-based probes (ABDA and DPA) have emerged as attractive tools for 1O2 detection, offering a promising alternative to conventional detection approaches. However, our results demonstrate that conventional ABDA/DPA analyses based on optical signal attenuation and mass-based assignment are not sufficiently specific. Non-1O2 oxidants in AOPs, including •OH, SO4•–, Fe(IV), and electron-transfer processes, produce similar optical responses and generate isomeric oxidation products that cannot be reliably distinguished from authentic 1O2-derived endoperoxides (ABDAO2 and DPAO2) by nominal mass matching or commonly used MRM transitions alone. To resolve this critical ambiguity, we establish reference-based LC-MS/MS fingerprints from well-characterized 1O2 generation systems, enabling discrimination of authentic 1O2-derived endoperoxides from structurally related oxidation products in AOPs. We further develop a semiquantitative framework that correlates endoperoxide formation with 1O2 generation, enabling semiquantitative assessment of 1O2 formation in oxidative systems. This work establishes a more defensible evidentiary framework for anthracene-probe-based 1O2 detection and mechanistic attribution, enabling more reliable identification of 1O2 involvement in oxidative systems relevant to water treatment.