Stability‐Indicating Ultrahigh‐Performance Liquid Chromatography Method for Quantification of Related Impurities in Tovorafenib Bulk Drug Using an Analytical Quality by Design Approach

Tovorafenib is a novel, selective, and potent Type‐II rapidly accelerated fibrosarcoma kinase inhibitor recently approved for the treatment of pediatric low‐grade glioma associated with B‐rapidly accelerated fibrosarcoma gene alterations. In this study, a novel, sensitive, and stability‐indicating ultrahigh‐performance liquid chromatography method was developed and validated for the quantification of related impurities in tovorafenib bulk drug using an analytical quality by design approach. Chromatographic separation was achieved on a reversed‐phase octadecylsilane silica column (Phenomenex Luna, 150 mm × 4.6 mm, 5.0 µm) under isocratic conditions using a mobile phase composed of 20 mM potassium phosphate buffer (pH adjusted to 2.5 with ortho phosphoric acid) and acetonitrile in a 70:30 volume ratio, at a flow rate of 1.0 mL/min. The injection volume was 10 µL, and detection was performed at 257 nm using an ultraviolet detector. The method was validated according to International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines Q2(R1) and demonstrated acceptable specificity, precision (relative standard deviation <0.5%), linearity (correlation coefficient = 1.000), accuracy (99.0%–99.9% recovery), sensitivity (limit of detection: 0.3 µg/mL; limit of quantification: 1.0 µg/mL), and robustness. The robustness of the method was carried out using Design Expert software, identifying flow rate, column temperature, and buffer pH as critical parameters. Robustness data were statistically validated using analysis of variance. Forced degradation studies conducted under acidic, basic, oxidative, thermal, photolytic, and neutral stress conditions confirmed the method's specificity and stability‐indicating capability, with peak purity passing in all the degradation conditions. This validated chromatographic method is suitable for routine use in quality control and quality evaluation of tovorafenib during pharmaceutical development and manufacturing.