Ultrahigh-performance liquid chromatography-ultraviolet absorbance detection-high-resolution-mass spectrometry combined with automated data processing for studying the kinetics of oxidative thermal degradation of thyroxine in the solid state

Abstract

Received 29 June 2014 Received in revised form 29 September 2014 Accepted 24 October 2014 Available online 30 October 2014 Keywords: Thyroxine Ultrahigh-performance liquid chromatography Electrospray ionization Orbitrap mass spectrometry Kinetics Drug degradation Bioinformatics 1. Introduction Levothyroxine (T4) is a well-known and widely used drug for the treatment of hypothyroidism. The therapy is usually based on substitution of the natural hormone by a long-term treatment with synthetic levothyroxine [1]. It is a narrow therapeutic index drug, for which individual dosage levels of patients need to be established over several weeks. Consequently, products differing in content of levothyroxine due to compound instability or problems in formu- lation can lead to wrong medication and potentially cause severe health problems [2,3]. ∗ Corresponding author. Tel.:+43 0 662 8044 5704; fax: +43 0 662 8044 5751. E-mail addresses: volker.a.neu@basf.com (V. Neu), chris.bielow@fu-berlin.de (C. Bielow), reinert@inf.fu-berlin.de (K. Reinert), c.huber@sbg.ac.at (C.G. Huber). http://dx.doi.org/10.1016/j.chroma.2014.10.071 0021-9673/© 2014 Elsevier B.V. All rights reserved. abstract Levothyroxine as active pharmaceutical ingredient of formulations used for the treatment of hypothy- roidism is distributed worldwide and taken by millions of people. An important issue in terms of compound stability is its capability to react with ambient oxygen, especially in case of long term com- pound storage at elevated temperature. In this study we demonstrate that ultrahigh-performance liquid chromatography coupled to UV spectrometry and high-resolution mass spectrometry (UHPLC-UV-HRMS) represent very useful approaches to investigate the influence of ambient oxygen on the degradation kinet- ics of levothyroxine in the solid state at enhanced degradation conditions. Moreover, the impurity pattern of oxidative degradation of levothyroxine is elucidated and classified with respect to degradation kinetics at different oxygen levels. Kinetic analysis of thyroxine bulk material at 100 ◦ C reveals bi-phasic degra- dation kinetics with a distinct change in degradation phases dependent on the availability of oxygen. The results clearly show that contact of the bulk material to ambient oxygen is a key factor for fast compound degradation. Furthermore, the combination of time-resolved HRMS data and automated data processing is shown to allow insights into the kinetics and mechanism of impurity formation on individual com- pound basis. By comparing degradation profiles, four main classes of profiles linked to reaction pathways of thyroxine degradation were identifiable. Finally, we show the capability of automated data process- ing for the matching of different stressing conditions, in order to extract information about mechanistic similarities. As a result, degradation kinetics is influenced by factors like availability of oxygen, stressing time, or stressing temperature, while the degradation mechanisms appear to be conserved.