Development of a bioanalytical method for quantification of methoxy-polyethylene glycol propionic acid polymer based on ultra-high performance liquid chromatography tandem mass spectrometry technique and micro-solid phase extraction to enhance sensitivity and greenness.

Revealing the potential of cellular cytotoxic and pharmacokinetics for methoxy‑polyethylene glycol propionic acid (M-PEG-PA) polymers is critical for evaluating the biocompatibility of PEG-based nanocarriers. M-PEG6-PA was employed as a target compound in this study. Cellular uptake of M-PEG6-PA polymers was first investigated by a green and selective ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay. After micro-solid phase extraction (μ-SPE), M-PEG6-PA and internal standard (IS) M-PEG5-PA were separated on an ACQUITY UPLC® BEH C18 (2.1 × 50 mm I.D, 1.7 μm) chromatographic column. Multiple reaction monitoring (MRM) of characteristic transitions at mass-to-charge ratio (m/z) 367.2 [M-H]- → 118.8 and m/z 323.2 [M-H]- → 75.1 were selected for quantification of M-PEG6-PA and M-PEG5-PA polymers, respectively. The established analytical method for quantitative analysis of M-PEG6-PA polymer demonstrated strong linear correlation within 0.01 to 1 μg/mL. This validated assay was subsequently applied to investigate cellular internalization of M-PEG6-PA in MCF-7 cell samples. The experimental results revealed minimal cytotoxic effects of M-PEG6-PA at exposure levels spanning 3 μg/mL to 3 mg/mL, with intracellular accumulation remaining below 0.03 % of administered doses in MCF-7 cell samples.