High-throughput UPC2-MS/MS for elucidating the molecular-weight-dependent excretion kinetics of PEG polymers with 10–17 subunits

Individual polyethylene glycol (PEG) oligomers’ in vivo fate is crucial for evaluating polymer-based therapeutic safety. Herein, a high-throughput UPC 2 -MS/MS method was developed for the oligomer-resolved excretion kinetics of PEG600 ( n  = 10–17) in rats. By optimizing supercritical CO 2 chromatography and utilizing ammonium adducts ([M+NH 4 ] + ), baseline separation of eight oligomers was achieved within 2.4 min, significantly enhancing efficiency for bioanalysis. This platform significantly enhances analytical capacity, enabling the efficient processing of large-scale biological sample sets required for mass balance studies. Application of this method revealed distinct molecular-weight-dependent elimination. Specifically, 72-h urinary recovery showed a complex non-linear relationship, peaking at n  = 10 (71.9%) and n  = 16 (72.7%). Simultaneously, total cumulative recovery (urine and feces) decreased progressively from >95% ( n  = 10, 11) to 75.3% ( n  = 17). This reduction suggests enhanced tissue sequestration or metabolic biotransformation for larger oligomers. This high-resolution profiling uncovers subtle elimination differences obscured in bulk analysis, providing critical pharmacokinetic insights for PEG excipients. Graphical abstract