Methoxy-polyethylene glycol amine (mPEG-NH₂), an important pharmaceutical excipient, has been extensively utilized in the field of biomedicine. To advance the development of mPEG-NH₂-related drug delivery systems, it is crucial to understand the safety profile and cellular pharmacokinetic behavior of mPEG-NH₂ polymers. In this study, a straightforward analytical assay based on ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) for quantifying mPEG-NH₂ in biological matrices was established and validated. Multiple reaction monitoring (MRM) transitions were selected for quantification of mPEG-NH₂ (mass-to-charge ratio, m/z 252.1 → 87.7) and octaethylene glycol (OH-PEG-OH) (mass-to-charge ratio, m/z 371.2 → 89.2). The UHPLC-MS/MS assay demonstrated excellent linearity within the concentration range of 0.01-10 μg/mL, with an R² value of 0.9996. Both intra-day and inter-day accuracies and precisions of the analytical method were within ± 9.19 %. This analytical assay was successfully applied to study the in vitro cellular toxicity and uptake behavior of mPEG-NH₂ in MCF-7 cells. The results indicate that high doses of mPEG-NH₂ may have potential toxicity to MCF-7 cells.
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