Determination of amphotericin B in rat blood plasma. Comparison of antifungal activity and pharmacokinetics profile of conventional amphotericin B and its conjugate with poly(ethylene glycol)

Abstract

New intravenous non-covalent conjugate of amphotericin B (AMB)-poly(ethylene glycol)(PEG) (M = 10 000 g mol–1) is described. This well water soluble conjugate contains 4 % (w/w) free AMB. A simple and sensitive HPLC-UV method was developed and validated for the quantification of free amphotericin B in rat plasma. A simple mobile phase consisting of 10 mM ammonium acetate (pH 3.60) and acetonitrile (56:44, v/v) was pumped at a flow rate of 0.3 ml min–1 through a reverse phase column maintained at 30 °C. Rifampicin was used as an internal standard (IS). Rapid sample preparation involved the addition of 500 µl acetonitrile-methanol mixture and 10 µl IS to 200 µl plasma to precipitate plasma proteins. Supernatant was evaporated to dryness under the stream of nitrogen and dissolved in 125 µl of the mobile phase and injected onto column. The procedures were validated within the linear concentration range from 0.06 to 2.5 µg ml–1 with good reproducibility and linear response (r2 = 0.9988). The method described is cost-effective and has necessary accuracy and precision for the rapid quantitative determination of AMB in rat plasma. A two-compartment model described the plasma drug concentration-time profiles.