A Rapid HPLC Method for the Simultaneous Determination of Amiodarone and its Major Metabolite in Rat Plasma and Tissues: A Useful Tool for Pharmacokinetic Studies

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed and validated in rat plasma and tissue (heart, liver, kidney and lung) homogenates for the determination of amiodarone and its primary metabolite (desethylamiodarone), using tamoxifen as internal standard. Chromatographic separation was achieved within less than 5 min on a LiChroCART Purospher Star C18 column (55 x 4 mm, 3 mu m). The mobile phase, consisting of phosphate buffer (50 mM) with 0.1% formic acid (pH 3.1)-methanol-acetonitrile (45:5:50, v/v/v), was pumped isocratically at a flow rate of 1.2 mL/min. The detection was conducted at 254 nm for all compounds. Calibration curves were linear (r(2) >= 0.995) in the range of 0.1-15 mu g/mL for amiodarone and desethylamiodarone. The limits of quantification were established at 0.1 mu g/mL for both analytes. The overall data of precision and accuracy were in accordance with international guidelines for bioanalytical method validation. Amiodarone and desethylamiodarone were extracted from rat matrices by a liquid-liquid extraction procedure and the mean recovery ranged from 59.9 to 97.6%. This novel HPLC method allows the fast and reliable determination of amiodarone and desethylamiodarone from several rat matrices (plasma, liver, kidneys, lungs and heart) and was successfully applied in a preliminary pharmacokinetic study.