Profiling polysorbate 80 components using comprehensive liquid chromatography-tandem mass spectrometry analysis.

Rationale: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant commonly used in pharmaceutical protein formulations and is composed of fatty acid (FA) esters of polyethoxylated sorbitan. However, commercial PS80 products contain substantial amounts of by-products. The development of simple and reliable methods for PS80 component analysis is challenging given the inherent heterogeneity.

Method: We developed a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to profile the components of PS80. Semi-comprehensive LC-MS/MS analyses of 11 subspecies in three commercial PS80 products were performed to estimate the average degree of polymerization of the ethylene oxide units (Avg-n) in the molecules. Furthermore, three subspecies (polyoxyethylene sorbitan monoester, polyoxyethylene isosorbide monoester, and polyoxyethylene monoester) were analyzed to estimate the composition ratios of the seven ester-bonded FAs present in PS80.

Results: The Avg-n values of five polyoxyethylene sorbitan esters (none, mono, di, tri, and tetra), three polyoxyethylene isosorbide esters (none, mono, and di), and three polyoxyethylene esters (none, mono, and di) were 26.5-30.6, 12.1-14.6, and 11.4-15.8, respectively. These values were comparable regardless of the number of ester-bonded FAs. Each product had a similar FA composition ratio regardless of the differences in the subspecies. However, the obtained C18:2 values were higher than those reported in the product certificates.

Conclusion: The proposed LC-MS/MS method evaluated the overall PS80 components, revealing the possibility of underestimation of ester-bonded linoleic acid using the conventional gas chromatography-mass spectrometry method. The similarity of Avg-n values and FA compositions among subspecies suggested the high reliability of these results, indicating that the presented approach may help in the quality control of PS80 formulations.