Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.