Quantitation of amphotericin B with use of high-pressure liquid chromatography

A chemical method for determination of concentrations of amphotericin B in serum and cerebrospinal fluid (CSF) is described. After extraction with methanol, the antibiotic was separated by reverse-phase, high-pressure liquid chromatography and quantitated by absorption at 405 nm. The lower limit of detection of this assay was 0.02 microng/ml. Relative standard deviations of less than 3.6% were noted for multiple determinations of sera containing 0.20 and 1.00 microng of amphotericin B/ml. No interfering peaks were found in extracts of serum or CSF from normal humans or in extracts of serum from patients treated with other drugs and antimicrobial agents, including 5-fluorocytosine. Comparison of the method with microbiological assays showed correlation coefficients of 0.90 and 0.83 for serum and CSF determinations, respectively. This chemical assay is very rapid (less than 30 min), sensitive, accurate, and specific and appears to be suitable for routine clinical use.