Detailed profiling of polysorbate 80 oxidative degradation products and hydrolysates using liquid chromatography-tandem mass spectrometry analysis.

RATIONALE: Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant that is commonly used to stabilize proteins in biopharmaceutical formulations. PS80 undergoes oxidative and/or enzymatic degradation. However, because PS80 is a complex mixture consisting of many constituents, comprehensive evaluations of its oxidative degradation products are difficult and insufficient. METHODS: Our previously reported comprehensive liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based method for PS80 effectively provides an overall profile of PS80 components under simple LC conditions. In this study, we attempted to shorten the analysis time. Furthermore, PS80 was oxidatively degraded in a solution containing histidine and iron, and the oxidative degradation products were evaluated using a modified LC/MS/MS method. In addition, enzymatically hydrolyzed PS80 samples were analyzed. RESULTS: We succeeded in shortening the analysis time from 70 to 20 min while maintaining the resolution of the PS80 components of the same selected reaction monitoring transition. Both the previously reported oxidative degradation products and the newly discovered products were successfully detected, and their composition ratios and changes over time were observed. Changes in the hydrolysates over time are shown in the analysis of the hydrolyzed PS80 samples. CONCLUSIONS: This study clearly showed the presence of changes in PS80 oxidative and/or enzymatic degradation products, including those previously unreported. These results demonstrate that a detailed profiling of PS80 degradation products can be performed using LC/MS/MS, which is less expensive and more generally adopted than high-resolution MS.

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.

Orally administered glycidol and its fatty acid esters as well as 3-MCPD fatty acid esters are metabolized to 3-MCPD in the F344 rat.

IARC has classified glycidol and 3-monochloropropane-1,2-diol (3-MCPD) as group 2A and 2B, respectively. Their esters are generated in foodstuffs during processing and there are concerns that they may be hydrolyzed to the carcinogenic forms in vivo. Thus, we conducted two studies. In the first, we administered glycidol and 3-MCPD and associated esters (glycidol oleate: GO, glycidol linoleate: GL, 3-MCPD dipalmitate: CDP, 3-MCPD monopalmitate: CMP, 3-MCPD dioleate: CDO) to male F344 rats by single oral gavage. After 30 min, 3-MCPD was detected in serum from all groups. Glycidol was detected in serum from the rats given glycidol or GL and CDP and CDO in serum from rats given these compounds. In the second, we examined if metabolism occurs on simple reaction with rat intestinal contents (gastric, duodenal and cecal contents) from male F344 gpt delta rats. Newly produced 3-MCPD was detected in all gut contents incubated with the three 3-MCPD fatty acid esters and in gastric and duodenal contents incubated with glycidol and in duodenal and cecal contents incubated with GO. Although our observation was performed at 1 time point, the results showed that not only 3-MCPD esters but also glycidol and glycidol esters are metabolized into 3-MCPD in the rat.