High-performance liquid chromatographic determination of amphotericin B in a liposomal pharmaceutical product and validation of the assay

Abstract

A validated high-performance liquid chromatographic method is presented to quantitate amphotericin B (AB) in a liposomal pharmaceutical formulation. The analysis is based on the chromatographic separation of AB and 1 -amino-4-nitronaphthalene (the internal standard) on a C18μBondapac reversed-phase column with a mobile phase consisting of a mixture of acetonitrile and 0.02M ethylenediamine tetra-acetic acid disodium salt at pH 5.0 (45:55, v/v). The chromatographic analysis time is less than 10 min, and the validation of the assay shows that it is selective, accurate, and linear for the concentration range of 2.50 to 7.50 μg/mL with a detection limit of 0.00500 μg/mL. The within-day and between-day relative standard deviation values are 1.26% (n = 18) and 1.25% (n = 8), respectively. The method described conforms to the validation of compendial methods used for finished pharmaceutical products in general and offers a reliable, quick, and cost-effective procedure for examining the consistency or quality-control analysis of AB in liposomal products. It can also be applied for the determination of AB in other nonliposomal lipid-based drug delivery systems that are on the market.