Study on the Forced Degradation Behaviour of Oteseconazole and Characterization of Its Degradants by LC-MS/MS.

Introduction: Oteseconazole is a novel tetrazole antifungal agent used to treat recurrent Vulvovaginal Candidiasis in women. Its mechanism of action is through the inhibition of cytochrome P450(CYP)51, thereby affecting the development and integrity of the fungal cell membrane. The main objective of the present work is to investigate the forced degradation behaviour of Oteseconazole, identify its degradants, and propose the fragmentation pathways of the degradants.

Methods: Good chromatographic separation of Oteseconazole was achieved using an HPLC System equipped with a PDA detector, and an X-Bridge Phenyl column (150 x 4.6mm, 5μm) with a mobile phase comprising acetonitrile and trifluoroacetic acid buffer (50:50) run in isocratic mode at 268 nm. For mass analysis, the HPLC system was connected to a SCIEX QTRAP 5500 mass spectrometer, operated in positive ion electrospray ionization interface mode.

Results: An accurate, specific, and time-efficient stability-indicating RP-HPLC method was developed for the estimation of Oteseconazole and its degradation products. The developed method was validated with Linearity in a range of 1.25μg/ mL - 7.5 μg/mL, accuracy (%RSD 0.06), system precision (%RSD 0.256), method precision (% RSD 0.49), LOD(0.3μg/mL), and LOQ(1μg/mL). Upon forced degradation studies according to ICH guidelines, Oteseconazole was found to be stable in photolytic, hydrolytic, and thermal conditions but degraded readily in acidic, alkaline, peroxide, and reduction environments.

Discussion: The developed method was robust and can be used in routine analysis to quantify Oteseconazole. The degradation behaviour of Oteseconazole was studied by performing forced degradation studies according to the ICH guidelines, and it was found to be stable in photolytic, hydrolytic, and thermal conditions, but degraded more readily in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS, and their fragmentation pathways were proposed.

Conclusion: Oteseconazole remained stable in photolytic, hydrolytic, and thermal conditions, but showed significant degradation in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS and characterized as 5-(4-chlorophenyl)-2-(2- (2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridine (acid impurity, DP1), sodium 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)phenolate (alkali impurity, DP2), 2-(2-(2,4-difluorophenyl)-1,1-difluroropropyl)-5-(4-hydroperoxyphenyl)pyridine (peroxide impurity, DP3) and 4-(6-2(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-tetrazol-1- yl)propyl)pyridin-3-yl)phenyl hydrogen sulfate (reduction impurity, DP4), and their fragmentation pathways were proposed. The developed method was accurate, specific, less time-consuming, and could be used in routine analysis for the quantification of Oteseconazole.