Amphotericin B determination in respiratory secretions by reversed-phase liquid chromatography.

Direct delivery of amphotericin B (AMB) to the respiratory tract may be an alternative to intravenous administration. The use of inhalation allows high AMB concentrations to be achieved at the site of infection. A reversed-phase high-performance liquid chromatographic method with a 30-mm-long column is described for assaying AMB in respiratory secretions obtained by bronchoaspiration (BAS) and bronchoalveolar lavage (BAL). Sample clean-up involved treatment with methanol (BAS) and solid-phase extraction onto Sep-Pak C18 cartridges (BAL). The mobile phase consisted of 2.5 mM Na2EDTA-acetonitrile (70:30, v/v). The retention time of AMB was 1.5 min. The range of the assay was from 0.1 to 5 micrograms/ml. The mean recovery was over 90% for both fluids. Within-day and between-day RSDs ranged from 3.10 to 11.87%. AMB in the BAS samples was stable for two days at 20-25 degrees C and for three months at -20 degrees C. The drug in the BAL fluid was stable for one day at 20-25 degrees C, seven days at 4 degrees C and for one month at -20 degrees C.