Optimization of LC-MS/MS Analytical Method for Trace Level Quantification of Potential Genotoxic Impurities in Siponimod Pure Drug and Formulations

Abstract

The generation of single or multiple genotoxic impurities during synthesis of siponimod should be avoided for production of safe formulation. Technically, complete elimination of genotoxic impurities was not possible and hence there is a need to propose an accurate method for trace level detection of genotoxic impurities.Method optimization studies were conducted by analysis standard solution in various method parameters. The results noticed in every varied method condition were tabulated for finalizing the appropriate conditions for analyzing siponimod. The optimized method consists of waters C18 (150 × 4.6 mm; 5 μm) column, ammonium acetate (0.02M) at pH 4.2 (fixed with 1 % formic acid) and methanol in 45:55 (v/v) at 0.5 mL/min flow rate. The mass analyser was operated in multiple reaction positive ion mode with characteristic mass transition at m/z of 517 (parent ion)and 213 (product ion) for siponimod, 434(parent ion) and 173 (product ion)for alcohol and 432(parent ion) and172 (product ion)for aldehyde impurity. No impurity or unwanted compounds detected in both LC chromatograms and mass spectra, confirming the method specificity.Validation of method for parameters including linearity, precision, recovery, ruggedness, and robustness yielded acceptable results. The method is suitable for assessing potential genotoxic impurities during the synthesis of siponimod and the manufacturing of pharmaceutical products.