Ammonium adduct-triggered MS³ detection and post-column infusion in UPC²-MS³: A green strategy for sensitive tissue distribution study of methoxy polyethylene glycol with 6 subunits in zebrafish

Short-chain polyethylene glycol derivatives, such as methoxy polyethylene glycol with 6 subunits (M‑PEG₆‑OH), are extensively utilized excipients and building blocks, yet their in vivo biodistribution and associated safety profiles remain insufficiently characterized. To address this knowledge gap, this study developed a novel, environmentally conscious analytical platform based on ultra‑performance convergence chromatography‑tandem mass spectrometry (UPC²‑MS³) for the sensitive quantification of M‑PEG₆‑OH in zebrafish tissues. A key methodological innovation involved the selective formation and detection of ammonium adducts ([M+NH₄]⁺) as precursor ions, coupled with a post‑column infusion strategy. This combined approach effectively mitigated ion suppression and significantly enhanced ionization efficiency, yielding an average 5‑fold increase in analytical signal response. The fully validated method achieved rapid and baseline separation of the analyte within 1.5 min. Its successful application to a tissue distribution study revealed distinct, time‑dependent accumulation profiles, with the highest levels observed in gill, followed by liver and muscle tissues. Furthermore, the methodology was rigorously evaluated using established metric systems, demonstrating excellent environmental sustainability (AGREE score = 0.62) and outstanding practical applicability (BAGI score = 72.5). This work provides crucial insights into the tissue‑specific disposition of short‑chain PEG derivatives and establishes a sustainable, high‑performance analytical framework for the safety evaluation of PEGylated compounds.