Development of a stability-indicating HPLC method for Lasmiditan and its process related impurities with characterization of degradation products by LC-MS/MS

The present investigation aimed to investigate a novel approach involving the utility of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS) for separation and characterization of very nominal quantities of degradation products (DPs) of lasmiditan. The process related impurities along with lasmiditan were resolved on Inertsil ODS-3 (250x4.6 mm, 5.0 mu m) column at room temperature using 0.1 M phosphate buffer with pH 3.6 and acetonitrile 65:35 (v/v) as mobile phase A, 0.1 M phosphate buffer with pH 3.6 and methanol in the ratio of 20:80 (v/v) as mobile phase B. The mobile phase solvent A and B were mixed at 50:50 (v/v) and the mixture was pumped isocratically at 1.0 mL/min and UV detection at 246 nm. The method shows sensitive detection limit of 0.003 mu g/mL for impurity 1, 0.008 mu g/mL, 0.005 mu g/mL for impurity 4, 0.008 mu g/mL for impurity 1 and 2 with calibration curve liner in the range of 25-150 mu g/mL and 0.025-0.15 respectively for lasmiditan and its studied impurities. The validation including system suitability, specificity, accuracy, recovery, ruggedness and robustness were noticed to be acceptable. The lasmiditan pure compound was subjected to stress studies as per guidelines of International Conference on Harmonization (ICH). The stress study results suggest that, lasmiditan was labile in acid, base and UV light conditions, whereas stable in thermal and peroxide conditions. A total of five degradation products (DPs) were formed and was characterized using fragmentation pattern as well as masses obtained on LC-MS/MS. The hitherto unknown DPs were identified 2,4,6-trifluoro-N-{6-[hydroxy(2,3,4,5-tetrahydropyridin-4-yl)methyl]pyridin-2-l}benzenecarboximidic acid (DP 1), N-[6-(2-ethylbutanoyl)pyridin-2-yl]-2,4,6-trifluorobenzamide (DP 2), 2,4,6-trifluorobenzamide (DP 3), 2,3-dihydropyridin-4-yl(pyridin-2-yl)methanol (DP 4) and 2,4,6-trifluoro-N-[6-(2-methylidenebutyl) pyridin-2-yl]benzamide (DP 5). The proposed method was successfully applicable for routine analysis of lasmiditan and its process related impurities in pure drug and formulations and also applicable for identification of known and unknown impurities of lasmiditan.