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 treatmen t of severe infections. It is used as amphotericin B-deoxycholate and in or der to reduce amphotericin B toxicity as lipid-formulated complex (liposoma l or colloidal dispersion). A sensitive and specific analytical method is p resented for the separation of lipid-complexed and plasma protein-bound amp hotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) v ia Bond Elut C-18. The protein-bound amphotericin B has a higher affinity t o the SPE material and is therefore retained, whereas the lipid-complexed a mphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification wa s performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detec tion (lambda =405 nm) and a mixture of acetonitrile-methanol-0.010 M NaH2PO 4 buffer (41:10:49, v/v) as mobile phase. The retention time for amphoteric in B under the given conditions was 6.7 min. The calibration curves were fo und to be linear (r greater than or equal to0.999) in two different ranges (5.0-0.50 mug/ml and 0.50-0.005 mug/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from ot her drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed. (C) 2001 Elsevier Science B.V. All rights reserved.