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 (UHPLCQ/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 MS 2 were performed to obtain as much structural information as possible. In addition, six unknown impurities were separated by semipreparative liquid chromatography and characterized using LC-SPE-NMR. Results The MS 2 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.
Nucleotide analogs (NAs) are widely used as antiviral and anticancer agents, but their isomers often exhibit distinct biochemical properties, posing challenges for precise analysis. Traditional separation methods are limited by low resolution and lengthy procedures. While ion mobility spectrometry-mass spectrometry (IMS-MS) offers rapid separation, its resolution is insufficient for structurally similar NA isomers. This study addresses the critical need for a high-resolution method to separate and quantify NA isomers, which is essential for drug development and quality control. We developed a host-guest chemistry approach using cyclodextrins (CDs) and metal ions to enhance IMS-MS separation of two NA isomer groups (2F-2DC/5F-5DC/5F-2DC and 2F-2DR/5F-5DR/5F-2DR). The proposed approach utilized β-CD to form binary complexes that resolved F-DC isomers (R p-p = 1.11) through non-covalent interactions, while ternary complexes incorporating Li + ions enabled separation of F-DR isomers via metal-coordination assisted molecular recognition (R p-p = 1.71). Theoretical calculations and non-covalent interaction analyses revealed distinct non-covalent binding sites and conformational variations, thereby elucidating the mobility separation mechanism at the molecular level. Quantitative analysis demonstrated high sensitivity LOD = 0.13–0.14 ng mL-1 for absolute quantification and LOD = 1:39–42:1 for relative quantification with recovery≥77 %, RSD ≤ 4.2 %). The method was successfully applied to drug purity testing and physiological solutions. This work introduces a novel strategy combining host-guest chemistry with IMS-MS to resolve NA isomers, overcoming limitations of traditional techniques. The molecular-level insights into CD-isomer interactions and the robust quantification framework advance the field of analytical chemistry for nucleotide-derived drugs, ensuring safer and more precise therapeutic applications.
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