ABSTRACT
RATIONALE: Hydroxychloroquine sulfate is effective in the treatment of malaria and 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 chromatography-quadrupole/time-of-flight-tandem mass spectrometry (UHPLC-Q/TOF/MS/MS), and the structures were characterized using liquid chromatography-solid-phase extraction-nuclear magnetic resonance (LC-SPE-NMR) spectroscopy. METHODS: An Agilent InfinityLad Poroshell HPH-C18 column (100 × 4.6 mm, 2.7 µm) was used. 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 to obtain as much structural information as possible. In addition, six unknown impurities were separated by semi-preparative liquid chromatography and characterized using 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 structures of the unknown impurities were confirmed using NMR. In addition, some possible 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.