An ultra-performance convergence chromatography tandem mass spectrometry strategy for quantification of monodisperse polyethylene glycol polymers coupled with multiple fragmentation to enhance sensitivity.

Accurate quantification of hydrophilic monodisperse polyethylene glycol (PEG) derivatives in complex biological matrices remains challenging due to substantial matrix interference and poor retention in conventional chromatographic systems. This study presents an environmentally sustainable analytical strategy based on ultra-performance convergence chromatography coupled with triple-stage mass spectrometry (UPC2-MS3) for the highly selective determination of HO-PEG8-OH in MCF-7 cell lysates. By harnessing the orthogonal separation mechanism of supercritical CO2 and the exceptional noise reduction capability of MS3 technology, the developed method achieves a rapid analysis time of 1.7 minutes while eliminating the need for sample pretreatment. The sustainability and practical applicability of the platform were systematically evaluated using a triple-metric framework, yielding favorable scores for GEMAM (7.166), AGSA (72.22), and CACI (75). Application of this sensitive approach to intracellular pharmacokinetic studies revealed an extremely low internalization rate of 0.0355% following 48 h exposure. Compared to existing methodologies, the UPC2-MS3 strategy offers a more robust, selective, and eco-friendly tool for elucidating the biological fate of monodisperse polymers in cellular systems.