Development and validation of HPTLC method for estimation of defactinib in bulk drug

Abstract Defactinib is an orally bioavailable and selective inhibitor of focal adhesion kinase (FAK), which is involved in multiple carcinogenic signaling pathways. The present study was undertaken to develop and validate a simple, rapid, and reliable high-performance thin-layer chromatography (HPTLC) method for the estimation of defactinib in bulk drug. Defactinib API was used as the working standard, and chromatographic separation was achieved on pre-coated silica gel 60 F254 HPTLC plates using toluene: methanol: glacial acetic acid in the optimized ratio of 8:2:0.5 (v/v/v) as the mobile phase. The chamber was saturated for 30 min, and the plate was developed to a solvent-front distance of 80 mm. The developed method was validated in accordance with the principles of ICH Q2(R2) guidelines with respect to linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness. The method exhibited good linearity over the concentration range of 1–8 µg/band, with a correlation coefficient (R²) of 0.99496. The intra-day and inter-day precision studies showed %RSD values below 2%, indicating satisfactory precision. Accuracy studies performed at 80%, 100%, and 120% concentration levels showed percentage recoveries within 98.5–99.5%. The calculated LOD and LOQ were 0.49 µg/band and 1.51 µg/band, respectively. Robustness studies demonstrated that minor deliberate variations in mobile-phase composition, chamber saturation time, and detection wavelength did not significantly affect the method performance. The developed HPTLC method provides a simple, accessible, and reliable approach for routine estimation and quality assessment of defactinib in bulk drug.