DETERMINATION OF AMPHOTERICIN-B IN HUMAN PLASMA BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING A SHORT OCTYL COLUMN

Amphotericin-B is a polyene antifungal antibiotic used for the treatment of severe systemic fungal infections. For effective treatment of urinary fungaria and the prevention of significant adverse-effects, monitoring the concentration of Amphotericin-B in biological samples of humans (ingesting the drug) is required. In this experiment, Amphotericin-B was isolated from plasma endogenous substances by adding 200-mu-L of acetonitrile in 800-mu-L of plasma. This mixture was vortex mixed, 20 mg of zinc sulfate and 10 mg of monobasic potassium phosphate was added to the mixture. This mixture was again vortex mixed and followed by centrifugation. The supernatant was filtered through a 0.45-mu-m membrane and a 100-mu-L aliquot of this solution was injected onto the chromatographic system. A short column of 60 mm x 4.6 mm packed with 3-mu-m octyl particles was used with an isocratic elution of 50/50, acetonitrile/0.01M KH2PO4 (v/v). The pH of the mobile phase mixture was adjusted to 3.5 with H3PO4. The intact drug molecule (parent drug) was monitored by a UV-visible detector at 410 nm and 0.10-0.005 A.U.F.S. The limits of detection of the method were 0.03-mu-g/mL for 100-mu-l injection volume at signal-to-noise ratio of 3.