Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: specific determination of oxidation products of thermally oxidized polysorbate 80.

The polysorbate species in polysorbate 80 (PS 80) have been characterized with (1)H NMR, LC-UV/MS and MS/MS. The MS was operated with both negative and positive electrospray ionization (ESI). PS 80 was found to contain mono- and di-fatty acid esters of polyoxyethylene (POE) sorbitan, POE isosorbide and of POE. In addition, the same species were also present without fatty acid esters. The polysorbate species were esterified with C12:0, C14:0, C16:1, C16:0, C18:2, C18:1 and C18:0 fatty acids. The main species were polysorbate esters of C18:1. The C18:2 polysorbate species were shown, by (1)H NMR, to have a large component of fatty acids with a conjugated double bond of ZE and/or EZ configuration. The positive ESI mass spectra of the polysorbate species displayed a specific in-source fatty acid fragment for each fatty acid ester. The mass chromatograms of the in-source fatty acid fragments were used to determine the degradation of specific polysorbate species in PS 80. The C18:2 polysorbate species were completely degraded after 8 weeks at 40°C under an atmosphere of air, while the main C18:1 polysorbate species were reduced to ca. 80-86% accompanied by an increase of short-chain POE C18:1 species. Polysorbate species esterified with C18:1-OH, C18:1 keto and C18:0 epoxy acids were found as degradation products of PS 80 stored at 40°C for 8 weeks under air. C18:1-OH, C18:1 keto and C18:0 epoxy acids were believed to be oxidation products of C18:1. With the present conditions, the positive ESI mass spectra of C18:1-OH and C18:0 epoxy polysorbate species displayed identical ions to the C18:2 polysorbate species due to a facile in-source loss of H(2)O from the protonated molecules. The presence of polysorbate esters of C18:1-OH and C18:0 epoxy acids were established using negative ESI MS. The presence of oxidized fatty acids in degraded PS 80 was also confirmed by saponification and extraction followed by negative ESI LC-MS analysis of the free fatty acids.