Development of a novel hplc method for the determination of axitinib in human plasma based on fluorimetric detection and precolumn derivatization with 7-chloro-4-nitrobenzofurazan: Application to a prototype pharmacokinetic study

Tyrosine kinase inhibitors (TKIs) are central to modern targeted cancer therapy. Axitinib, a highly selective oral TKI, primarily inhibits angiogenesis. This study aimed to develop a fast, reliable, and simple HPLC method with fluorescence detection to measure axitinib in human plasma. Sample preparation was performed using a simple and efficient liquid-liquid extraction (LLE) procedure. Axitinib was isolated from alkalinized plasma using chloroform, followed by evaporation of the organic phase. Prior to analysis, axitinib was derivatized with 7-chloro-4-nitrobenzofurazan (NBD-Cl). Separation was performed on a reversed-phase C18 column (2.6 µm, 4.6 mm × 150 mm) at 30 °C under isocratic conditions, using a mobile phase of acetonitrile and 0.1% orthophosphoric acid (75:25 v/v) at 0.9 mL min⁻¹. Fluorescence detection was set at 330 nm excitation and 405 nm emission. Axitinib eluted at 3.15 ± 0.02 minutes. The method showed excellent linearity from 0.01 to 30 ng mL⁻¹ (r = 0.9998), with LOD and LOQ of 0.003 ng mL⁻¹ and 0.01 ng mL⁻¹, respectively. Precision, expressed as RSD, did not exceed 2.65%. Validation confirmed compliance with EMA bioanalytical guidelines. The method was successfully applied in pharmacokinetic studies, enabling determination of AUC₀–t, AUC₀–∞, Cmax, Tmax, and t½. Thanks to its sensitivity and robustness, this HPLC fluorescence method is suitable for routine quantification of axitinib in human plasma and can be applied in bioavailability and bioequivalence studies.