High throughput analysis of methoxy polyethylene glycol polymers with 6 subunits by UPCC-MS3 coupled with multiple fragmentation to improve sensitivity

A green and highly selective Ultra-Performance Convergence Chromatography with MS 3 (UPC 2 -MS 3 ) method was developed for the quantification of M-PEG₆-OH in rat plasma. • The MS 3 detection provided exceptional specificity by effectively eliminating matrix interferences and offered a superior signal-to-noise ratio compared to conventional MRM. • Utilizing supercritical CO₂ as the primary mobile phase enabled a 1.5 min analysis, significantly reducing organic solvent consumption and aligning with green chemistry principles. • The fully validated method was successfully applied to characterize the pharmacokinetic profile of M-PEG₆-OH in rats. The quantification of hydrophilic polyethylene glycol (PEG) derivatives in biological matrices is often hindered by poor chromatographic retention and severe matrix effects in conventional reversed phase systems. This study addresses these challenges by developing a novel, green analytical platform integrating ultra-performance convergence chromatography (UPCC) with triple mass spectrometry (MS 3 ) detection. The method leverages the orthogonal separation mechanism of UPCC to achieve superior resolution within an ultra-short 1.5 min analytical cycle. Notably, the MS 3 acquisition strategy effectively eliminated background chemical noise, resulting in a 4.7-fold enhancement in the signal to noise ratio compared to standard MRM modes at the lower limit of quantification (LLOQ) level. The methodology was rigorously validated according to FDA guidance (adopted from the ICH M10 guidelines) over a range of 10–1000 ng/mL, demonstrating an LLOQ of 10 ng/mL. Quantitative greenness evaluation via AGREE (0.75) and BAGI (77.5) metrics confirmed the environmental sustainability of the approach. Successfully applied to a pharmacokinetic study in rats, this robust UPCC-MS 3 platform provides a high performance, compliant, and ecofriendly tool for the biological disposition assessment of PEG based therapeutics. In conclusion, the established platform successfully harmonizes high-throughput bioanalytical efficiency with green chemistry principles, demonstrating outstanding technical superiority and structural compliance for seamless integration into standardized laboratory workflows within the pharmaceutical industry. Download: Download high-res image (260KB) Download: Download full-size image