Identification of 5-methylisoxazole-4-carboxylic acid and 4-(trifluoromethyl)aniline degradation products for leflunomide by high-performance thin-layer chromatography coupled with high-resolution mass spectrometry

This study details the developed high-performance thin-layer chromatographic (HPTLC) method for estimating the active pharmaceutical ingredient of leflunomide and its formulation. HPTLC method was developed using pre-coated TLC plates (Merck) with silica gel G 60F(254). The optimized mobile phase was toluene-chloroform-ethanol (4:4:2, V/V), having UV detection at 266 nm. Forced degradation study was carried out using various stressors like acidic, alkaline, hydrolytic, oxidative, photolytic and thermal conditions (International Council for Harmonisation [ICH], Q1A [R2]). Leflunomide was susceptible to degradation in acidic, alkaline and neutral stressors, whereas minor degradation was observed in oxidative, photolytic and thermal stressors. Degradation products of acidic and alkaline conditions were characterized by high-resolution mass spectrometry (HR-MS). Degradation products identified after stress studies were 4-(trifluoromethyl)aniline and 5-methylisoxazole-4-carboxylic acid. Linearity studies were performed in the range of 100-600 ng/band. The parametric statistics of the calibration curve was found to be 0.9988. The limit of detection and limit of quantification values were found to be 4.35 ng/band and 13.19 ng/band. The developed method was studied as per the ICH Q2 (R1) guidelines for different parameters like linearity, accuracy, precision, specificity and robustness. Leflunomide in tablet dosage form was successfully analyzed using the described approach.