A VALIDATED LC-MS/MS METHOD FOR PHARMACOKINETIC STUDY OF AFATINIB IN HEALTHY RABBITSAbstract
A liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) method was developed for quantification of Afatinib in rabit plasma employing Solid Phase Extraction (SPE) technique. Developed method was validated for specificity, precision, accuracy, recovery, and stability characteristics. Chromatographic separation was achieved on Gemini, 5µ C18, 50 × 4.60 mm with 30:70 v/v of 10 mM ammonium acetate in water: Organic Mixture (methanol : acetonitrile, 80:20% v/v) as an isocratic mobile phase with a flow rate of 1.0 ml/min. the developed LC-MS method was applied to assess pharmacokinetics parameters of afatinib (20 mg) tablet in healthy rabbits. Six Male albino rabbits weighing 2.0 – 2.5 kg were randomly selected for the pharmacokinetic study. The tablets were administered to rabbits using a balling gun. Blood samples (0.6 ml) were withdrawn from the marginal ear vein before dosing (zero time) and at time intervals of 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 20, 24, 36 and 48 hr after administration. Plasma was separated by centrifugation at 5000 rpm for 10 min and the plasma concentrations of afatinib at different times were determined by LC-MS/MS. Pharmacokinetic parameters was calculated. Afatinib showed Tmax of 3.833 ± 0.752 and mean Cmax, AUC0®t and AUC0®a for Test formulation is 675.83 ± 22.031, 6213.417 ± 257.922 and 6474.61 ± 258.45 respectively.