Abstract: Ruan Hao (Orcid ID: 0000-0002-7096-0635) A study of impurities in the repurposed COVID-19 drug hydroxychloroquine sulfate by UHPLC-Q/TOF-MS and LC-SPE-NMR Donghai Xu a, Fangfang Pan b, Hao Ruana, b, *, Nan Sun a, ** a Zhejiang University of Technology, Hangzhou 310014, China b Zhejiang Institute for Food and Drug Control, National Medical Product Administration Key Laboratory for Core Technology of Generic Drug Evaluation, Hangzhou 310052, China * Corresponding author. Zhejiang University of Technology, Hangzhou 310014, China. ** Corresponding author. E-mail addresses: clare_ruan@163.com (H. Ruan), sunnan@zjut.edu.cn (N. Sun). ABSTRACT Rationale: Hydroxychloroquine sulfate is effective in the treatment of malaria, autoimmune diseases, and as an antiviral drug. However, unreported impurities are often detected in this drug, which pose a health risk. In this study, the structures of hydroxychloroquine and six unknown impurities were analyzed using ultra-high performance liquid chromatographyquadrupole/time-of-flight tandem mass spectrometry (UHPLC-Q/TOF MS), and the structures were characterized using liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy (LC-SPE-NMR). Methods: The column was an Agilent InfinityLad Poroshell HPH-C18 (100 mm × 4.6 mm, 2.7 µm). For the analysis of hydroxychloroquine and six unknown impurities, the mobile phase was 20 mM ammonium formate aqueous solution and methanol/acetonitrile (80:20, v/v), using gradient elution. Full-scan MS and MS2 were performed in order to obtain as much structural information as possible. Additionally, six unknown impurities were separated by semi-preparative liquid chromatography and characterized by LC-SPE-NMR. Results: The MS2 fragmentation patterns of the impurities were investigated, leading to more structural information and an understanding of the fragmentation pathways of the impurities. The unknown impurities’ structures were confirmed by NMR. In addition, some possible This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/rcm.9358 This article is protected by copyright. All rights reserved. pathways of the formation of the impurities in the drugs were outlined, and these impurities were found to be process impurities. Conclusions: Based on the identification and characterization of these impurities, this study also describes the cause of the production of the impurities and provides insights for companies to improve their production processes and a scientific basis for the improvement of the related pharmacopoeias. Graphical Abstract Keywords: hydroxychloroquine, impurities, UHPLC-Q/TOF-MS, LC-SPE-NMR, characterization This article is protected by copyright. All rights reserved.