An internally-standardized assay for amphotericin B in tissues and plasma

A high-performance liquid chromatographic (HPLC) method with p-nitrophenol as internal standard is described for the rapid analysis of amphotericin B recovered by methanolic extraction from tissues and plasma. Programmed, gradient elution of the ODS column was used with detection by tungsten light at 388 nm. Standard curves were derived based on the peak height ratios. The lowest reproducible limit of the assay was 0.04 micrograms/ml with plasma. The extraction and chromatographic procedures recovered 53-71% of the amphotericin B from each of these sources. The coefficient of variation of the recovery ratios was less than 18% from plasma over a range of concentrations of amphotericin B from 0.08 to 10.0 micrograms/ml. Recovery from tissues, studied over a narrower concentration range, showed a similar degree of precision. Variations in precolumns apparently resulting in selective binding of the amphotericin B were found to have a systematic but important influence on recovery efficiency. No substances were detected which interfered with the assay procedures as described. By incorporating an internal standard we have enhanced the reliability and flexibility of the HPLC assay for amphotericin B especially for assay of tissues.