Brefeldin A (NSC 89671), a macrolide isolated from Penicillium brefeldianum, is undergoing preclinical evaluation as an antitumor agent at the National Cancer Institute. A specific assay for brefeldin A was developed involving its extraction from plasma into diethyl ether followed by derivatization with heptafluorobutyrylimidazole prior to gas chromatography with electron capture detection. The lowest concentration of brefeldin A determined with acceptable reproducibility in 50 micro l of plasma was 0.1 micro g/ml (0.36 micro M). This assay has been utilized to investigate brefeldin A plasma pharmacokinetics in mice. However, due to the rapid clearance of the drug by mice (102 ml/min/kg) and dogs (53 ml/min/kg), plasma levels could only be monitored for approximately 60 min following bolus i.v. administration of maximum tolerated doses (mouse approximately 26 mg/kg; dog approximately 8 mg/kg). Thus, alternate approaches were sought in an effort to develop a more sensitive method. Using reversed-phase HPLC with UV detection, an assay for the quantitation of brefeldin A in biological fluids was developed. The method employed isolation of the analyte by liquid/liquid extraction with tert-butyl methyl ether. Following evaporation of the organic solvent and reconstitution, the analyte was separated by reversed-phase HPLC and detection by UV absorption at 215 ng. The lower limit of quantitation for brefeldin A in a 100 micro l sample of plasma was 0.1 micro g/ml. While this did not represent any improvement in sensitivity over the GC method, the HPLC assay permitted specific determination of the drug and was technically more convenient than the GC assay. Employing LC/thermospray MS, the lowest concentration of brefeldin A quantifiable in 50 micro l of plasma was 0.005 micro g/ml (0.014 micro M). The dynamic linear range of the assay extended from 0.005 to 0.500 micro g/ml, with a correlation coefficient typically 0.99 or greater. This represents an improvement in sensitivity by about a factor of 20 compared to previous methods. Validation and application of the method are currently in progress.
