Improved assay for serum amphotericin-B by fast high performance liquid chromatography

Abstract A selective and sensitive method for the determination of amphotericin B in dog serum, after deproteinization, using high performance liquid chromatography HPLC with UV detection at 382 nm was developed. After protein precipitation with methanol, 5 μl of the supernatant was injected onto a reversed phase C18 column (1.5μm, 33 × 4.6 mm ID), with a mobile phase composed of 0.05 M sodium acetate - acetonitrile - methanol (40:30:30, v/v). The analytical recovery of amphotericin B in serum was 92.30%. The linearity of the assay method was verified up to 15 μg/mL of amphotericin B. The assay was reproducible with satisfactory intra-day and inter-day coefficients of variations (CV < 3%). The sensitivity of the method was 0.625 ng of amphotericin B for serum. The method was successfully employed for the pharmacokinetic analysis of amphotericin B in dogs following intravenous administration of liposomal amphotericin B (AmBisome).