An analytical method using HPLC coupled with a charged aerosol detector (CAD) and a mass selective detector (MSD) was developed to characterize the non-ionic surfactant polysorbate 80 (PS 80). The molecular structure and heterogeneous composition due to isomers and various lengths of PEG-chains make it difficult to develop sensitive and specific analytical methods. Hence, there is only limited knowledge about the stability and purity of this compound. Polysorbate 80 does not possess any chromophore, thus UV detection is not applicable. Therefore, CAD and MSD have been used for determination. The aim of this study was to characterize polysorbate 80 and to examine its stability at pH 1.0 and 37 degrees C simulating harsh gastric conditions. It was shown that this surfactant is liable to degradation under these conditions. Within 8 h monoesters of PS 80 were hydrolyzed to an extent of 9.5% (+/- 3.0%), whereas incubation in water did not result in any detectable degradation. Furthermore, we demonstrated that HPLC-MS is a suitable technique to investigate ethoxylated compounds like polysorbates.