Development and validation of a novel analytical method for related substances of resmetirom and identification of new degradation products

Background: Resmetirom (MGL-3196), a highly selective thyroid hormone receptor β agonist, is indicated for treating adults with noncirrhotic nonalcoholic steatohepatitis (NASH) exhibiting moderate to advanced liver fibrosis. Related substances are critical factors that directly impact the safety and efficacy of the active pharmaceutical ingredient (API). Therefore, the quantitative detection of related substances in resmetirom is of particular importance. Methods: This study established a reverse-phase high-performance liquid chromatography (RP-HPLC) for the separation of resmetirom and its related substances. The analysis was performed using an Agilent 5 HC C18 (2) column (250 mm × 4.6 mm, 5 µm) at 35 ℃, with detection at 220 nm. A gradient elution method was employed, achieving effective separation of all impurities within a 45-minute runtime. The final HPLC method utilized with a mixture of H2O/ACN (90/10, v/v) containing 0.045% TFA as mobile phase A, and ACN as mobile phase B. The gradient elution proceeded as follows: 0~2 min, 10% B→ 10% B; 2~20 min, 10% B→ 50% B; 20~30 min, 50% B→ 54% B; 30~35 min, 54% B→ 54% B; 35~36 min, 54% B→ 10% B; and 36~45 min, 10% B→ 10% B. The rate of flow was 1.0 mL/min. A 10 µL volume was used for the sample injection. This is a provisional file, not the final typeset article Results: The method was validated according to International Conference on Harmonization (ICH) guidelines, and the results demonstrated that it possesses good specificity, stability, linearity, precision, repeatability, and robustness. Furthermore, a new degradation product, designated as imp-A, was generated under alkaline degradation conditions in this study. Its chemical structure was elucidated using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and this compound has been reported for the first time. Conclusion: The RP-HPLC method established in this study is capable of accurately and reliably separating and detecting resmetirom and its related substances. The method is simple to operate and yields dependable results, providing a practical and efficient analytical tool for the quality control of resmetirom and its formulations. This contributes to ensuring the safety and efficacy of the drug product.