Introduction. Research interest in orphan drugs has been growing annually. Consequently, new research studies and developments in quality control for this class of medicinal products are required. Given the relevance of this issue, the aim of this study is to investigate the fragmentation mechanism (FM) of the prospidium chloride (PrsCl₂) molecule under electrospray ionization (ESI) to develop new quality control methods using the ESI-HPLC-MS system. Materials and Methods. Prospidium chloride substance (PrsCl₂, Mr=500.3). Orbitrap Exploris 120 mass spectrometer, equipped with a Vanquish liquid chromatograph (Thermo Scientific, USA) and an Acclaim™ RSLC 120 C18 analytical column from Dionex Bonded Silica Products (Thermo Scientific, USA). Sample injection volume: V=5 µL; total analysis time: t=19 minutes. Studies were conducted with aqueous solutions at a concentration of 0.05 mg/mL. Results. The study describes the FM of singly and doubly charged ions formed from the active pharmaceutical ingredient (API): m/z=445,1819 [M – Cl^-]⁺, m/z= 409,2129 [M – 2Cl^- – H⁺]⁺, m/z=205,1101 [M – 2Cl^-]²⁺. However, as demonstrated, these ions may originate not only from the API but also from related impurities. This is critical to consider, as the presence of related impurities may overestimate quantitative analysis results. The study also highlights the FM of ions presumably formed from PrsCl₂ derivative compounds: m/z=373.2365 [M - 3Cl^- - 2H]⁺, m/z=187.1219 [M - 3Cl^- - H]⁺², m/z=169.1336 [M – 4Cl^- - 2H]²⁺. The study results can contribute to the development of a method for determining the purity of the substance.

Conclusion. The demonstrated FM of the PrsCl₂ molecule and its derivatives during chromatographic-mass spectrometric analysis enables the development of methods for qualitative and quantitative analysis, as well as the determination of the purity of the PrsCl₂ substance.

Funding: This research was supported by a grant from the RUDN University Strategic Academic Leadership Programme No. 033320–2-000.