Development and validation of a Liquid Chromatography–mass Spectrometry Method for Determination of Amphotericin B in Rabbit Tears and its Application to Ocular Pharmacokinetic Study

Abstract

A rapid and sensitive LC-MS-MS method was developed for the quantification of amphotericin B in rabbit tears using natamycin as internal standard. The analyte and internal standard were extracted from the tear sample using a solid phase extraction method. Chromatographic separation was achieved on a Phenomenex Luna 3 µm CN column (100 mm × 2 mm, 3 µm) using 3.5 mM ammonium acetate (pH 4): methanol (10 : 90) as mobile phase. The assay was validated with a linear range of 0.1 – 3.2 µg mL-1 for amphotericin B using 10 µL of tear sample. The intra- and inter-day assay precision ranged from 2.49 to 4.37% and 2.17 to 5.59%, respectively, and intra- and inter- day assay accuracy was between from 0.27 to 3.32% and -0.51 to 3.72%, respectively. The method was successfully applied to the pharmacokinetic studies of amphotericin B eye drops in rabbit tears.