title: Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis.
abstract: The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality control of newly developed and well-established active pharmaceutical ingredients (APIs). The United States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS absorption detection to determine organic impurities in the drug substance. The impurity profile of the first USP method ("Procedure 1") has already been extensively studied, however for the second method ("Procedure 2"), which exhibits a significantly different impurity profile, no wholistic structural elucidation of impurities has been performed yet. Applying minor modifications to the chromatographic parameters of USP "Procedure 2" and using various comprehensive structural elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted in the characterization of 24 compounds previously unknown to literature and the introduction of two new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified major impurities as well as neutral loss fragmentation patterns of many characterized impurities were discussed.
authors.affiliation: SANDOZ GmbH, Biochemiestraße 10, A-6250 Kundl/Tirol, Austria; Department of Molecular Biology, Division of Chemistry and Bioanalytics,University of Salzburg, Hellbrunner Straße 34, A-5020 Salzburg, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Ruggenthaler
authors.affiliation: SANDOZ GmbH, Biochemiestraße 10, A-6250 Kundl/Tirol, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Grass
authors.affiliation: SANDOZ GmbH, Biochemiestraße 10, A-6250 Kundl/Tirol, Austria.
authors.forename: W
authors.initials: W
authors.lastname: Schuh
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics,University of Salzburg, Hellbrunner Straße 34, A-5020 Salzburg, Austria.
authors.forename: C G
authors.initials: CG
authors.lastname: Huber
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics,University of Salzburg, Hellbrunner Straße 34, A-5020 Salzburg, Austria. Electronic address: roland.reischl@sbg.ac.at.
authors.forename: R J
authors.initials: RJ
authors.lastname: Reischl
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004340:Drug Contamination; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013974:Thyroxine / Q000032:analysis* / Q000138:chemical synthesis*
chemical_list: D013974:Thyroxine
keywords: HDX; LC-MS; Levothyroxine; On-line H/D Exchange; Pharmaceutical impurities; Thyroid hormone