Direct-Injection ESI-MS/MS Method for Studying Fragmentation Pathways of Prospidium Chloride

Objective: Investigation of the ionization and fragmentation behavior of precursor ions enables improvement of qualitative analytical approaches for pharmaceutical substances without using chromatographic separation. Identification of diagnostic product ions additionally facilitates the development of quantitative mass‑spectrometric methods based on MRM/SRM transitions and expands libraries of diagnostic mass ions for the studied drugs. Materials and Methods: Mass spectra of precursor and product ions of prospidium chloride were obtained using a High‑Performance Liquid Chromatograph—Triple Quadrupole Mass Spectrometer LCMS‑8050 equipped with electrospray ionization (ESI) and direct sample introduction (DIP). Results: Five precursor ions at m/z 187.2, 205.2, 373.3, 409.2 and 740.2 were detected and characterized. The most intense precursor ions at m/z 205.2 and 409.2 were selected for collision‑induced dissociation in the collision cell. MS/MS spectral analysis enabled elucidation of fragmentation pathways and the identification of a diagnostic product ion at m/z 164.4. Conclusion: The precursor ions reported here, described for the first time, can be used to broaden existing normative documentation for prospidium chloride by providing a non‑chromatographic method for authenticity testing. Elucidation of fragmentation mechanisms reveals diagnostic product ions that retain core structural elements of the prospidium chloride molecule and are suitable for development of quantitative mass‑spectrometric assays.