Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution mass spectrometry, on-line H/D exchange and UV/Vis absorption

[Display omitted] •Use of high resolution mass spectrometry and on-line H/D exchange for the impurity profiling of an important active pharmaceutical ingredient.•Characterization of 25 previously unknown impurities of liothyronine sodium.•Implementation of a wholistic thyroid hormone impurity classification scheme.•Discussion of the CID fragmentation mechanisms of characterized liothyronine sodium impurities. For the first time, a comprehensive investigation of the impurity profile of the synthetic thyroid API (active pharmaceutical ingredient) liothyronine sodium (LT3Na) was performed by using reversed phase HPLC and advanced structural elucidation techniques including high resolution tandem mass spectrometry (HRMS/MS) and on-line hydrogen-deuterium (H/D) exchange. Overall, 39 compounds were characterized and 25 of these related substances were previously unknown to literature. The impurity classification system recently developed for the closely related API levothyroxine sodium (LT4Na) could be applied to the newly characterized liothyronine sodium impurities resulting in a wholistic thyroid API impurity classification system. Furthermore, the mass-spectrometric CID-fragmentation of specific related substances was discussed and rationalized by detailed fragmentation pathways. Moreover, the UV/Vis absorption characteristics of the API and selected impurities were investigated to corroborate chemical structure assignments derived from MS data.