Resolving compositional heterogeneity in commercial high-molar-mass poloxamers by orthogonal and complementary...

Commercial poloxamers exhibit compositional heterogeneity, yet the minor species contributing to this heterogeneity are often incompletely resolved and therefore treated as a single impurity fraction. This limits accurate analytical characterization of pharmaceutical-grade materials and complicates the analysis of structure-property relationships. In this study, we establish an analytical approach that combines mixed-mode liquid chromatography, reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) with multiangle light scattering and refractive index detection, complemented by MALDI-TOF mass spectrometry and ¹H NMR spectroscopy, to separate, identify, and quantify minor side products in commercial high-molar-mass poloxamers. We identified poly(propylene oxide) (PPO)-poly(ethylene oxide) (PEO) diblock copolymers and PEO homopolymer as the principal side products, and determined the amount of PEO homopolymer at levels as low as ∼0.7 wt percent. Additionally, RPLC further resolved the compositional substructure within the PPO-PEO diblock population, revealing a distribution in the PPO block length that was not resolved by SEC alone. Comparison of commercial poloxamer 407 samples from different manufacturers showed that, among the heterogeneity parameters resolved here, differences in the PPO-rich character of the main PEO-PPO-PEO triblock population were most plausibly associated with the observed variations in critical micelle concentration and hydrodynamic radius of the micelles, whereas the quantified amount of PEO homopolymer had little effect under the conditions studied. Overall, the results show that combining analytical techniques enables comprehensive characterization of poloxamers by separating, identifying, and quantifying synthesis-derived side products, while also revealing the heterogeneity parameters most plausibly associated with micellization behavior.