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Objective: Developing and validating a stability-indicating method for everolimus by HPTLC and depiction of degradation product of in alkaline conditions by LC-MS. Methods: The chromatographic separation was performed on aluminium plates pre-coated with silica gel 60 F254 as the stationary phase using Toluene: Methanol: Ethyl Acetate (6:2:2v/v/v) as the mobile phase. The evaluation was carried out at 277 nm. For the developed stability indicating method, the ICH Q2 (R1) guidelines were used for validation. Stress degradation studies like hydrolysis under different pH conditions, photolytic degradation, thermal degradation and oxidative degradation as per ICH Q1A (R2) and Q1B guidelines were performed. LC-MS analysis was carried out for the standard everolimus and its alkaline degradation sample using TOF analyser and the degradation pathway was proposed for each degradation product. Results: The Rf value of everolimus was found to be 0.63±0.03. The response was quite linear over the concentration range of 100-500 ng/band, with the regression coefficient value of 0.9921. Under alkaline hydrolytic conditions, everolimus was analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). The Retention Time (RT) and prominent mass fragmentation (m/z) for the everolimus standard were observed at 9.46 min with m/z values of 980.56 and 908.54. For the degradation products, DP-1 showed an RT of 8.88 min with m/z values of 349.23, 403.24, 574.33, and 646.35, while DP-2 exhibited an RT of 9.10 min with m/z values of 926.55, 614.32, and 542.30. These data were used to propose the structures of the degradation products. Conclusion: The proposed method can conveniently be applied for quantitative analysis of everolimus on routine basis and for stability testing under different stress environments.
Damle et al. (Sun,) studied this question.