High-throughput and green UPCC-MS/MS assay for simultaneous quantification of 20 oligomers of PEG1K coupled with ammonium adduct and post-column infusion strategies to enhance sensitivity.

This study presents an innovative analytical approach utilizing ultra-performance convergence chromatography tandem mass spectrometry (UPCC-MS/MS) for the systematic separation and quantification of 20 oligomers (n = 9-28) of polyethylene glycol 1000 (PEG1K) in rat urine. A key innovation involves the use of ammonium adducts ( [M+NH₄]⁺ and [M + 2NH₄]²⁺) as precursor ions, significantly enhancing sensitivity and selectivity, particularly for higher oligomers (n > 21), where doubly charged species predominated. Coupled with a post-column compensation strategy, ionization efficiency was improved by 1-10-fold. Chromatographic separation was achieved under isocratic conditions using a green mobile phase based on supercritical CO₂ with a methanol-formic acid co-solvent, enabling efficient baseline resolution within a short runtime while minimizing organic solvent consumption. The method was validated per FDA guidelines, demonstrating excellent linearity, precision, and accuracy. Its applicability was confirmed through excretion kinetics studies in rats tail vein injection of PEG1K, revealing rapid renal clearance with 23% of the dose excreted within 4 h and cumulative excretion reaching 64% at 48 h. Green analytical chemistry metric assessments (BAGI and GEMAM) highlighted the method's superior environmental profile. This work establishes a sensitive, efficient, and sustainable platform for polymer pharmacokinetic research.