Studies on Amphotericin B Pharmacokinetics (1) Determination of Amphotericin B by High-Performance Liquid Chromatography

Amphotericin B (AMPH) is widely used in the treatment of fungal infectious disease. However, the relationship of the pharmacokinetics to the physicochemical properties and the therapeutic effects have not yet been clarified. To study the pharmacokinetics of AMPH, we developed a method to determine the concentration of AMPH in serum, feces and bile by high-performance liquid chromatography (HPLC). In the pretreatment, the sample was loaded on a Sep-pak cartridge minicolumn, which was successively washed with water, 30% methanol and acetonitrile; then AMPH was eluted with 10% DMSO in methanol from the minicolumn and injected into HPLC. Chromatography was performed on a reversed phase radial-PAK cartridge 8 MBC18 (10μm, Waters). The mobile phase was methanol/0.004M ethylenediaminetetraacetate (85/15). Flow rate was 2.0ml/min and detection wavelength was 405nm. Peak height was measured to construct the standard curve. In the chromatographic system, AMPH was eluted at a retention time of 4.2min. Interfering peaks were removed from the sample by the minicolumn pretreatment. The detection limit was 0.02μg/ml in serum, and a very small quantity of sample was required. This method can be used for analysis of various samples (such as tissue homogenates).