Brefeldin A (BFA) (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 BFA was developed involving its extraction from plasma into diethyl ether followed by derivatization with heptafluorobutyry-limidazole prior to gas chromatography (GC) with electron capture detection (ECD). This assay has been utilized to investigate BFA plasma pharmacokinetics in mice. However, using reversed-phase HPLC with UV detection, an assay with greater technical convenience for the quantitation of BFA in biological fluids was then developed. Although no more sensitive than the GC method, this new method employed simple isolation of the analyte by liquid/liquid extraction with tert-butyl methyl ether, evaporation of the organic solvent, reconstitution, and separation by reversed-phase HPLC with UV detection. However, failure to identify an acceptable injectable formulation has impeded development of BFA as a clinical candidate. Among several water-soluble BFA derivatives that have been prepared, breflate (NSC 656202) was selected for evaluation as a potential prodrug. Its solubility and stability in aqueous vehicles are more than adequate for parenteral delivery and a clinical formulation has been developed. The plasma pharmacoki-netics of breflate was investigated in the mouse and dog using an HPLC assay which permits concurrent quantitation of the prodrug and BFA. A continuous i.v. infusion schedule was investigated in dogs, and although the plasma levels of BFA could be determined, plasma levels of the prodrug remained below the limit of detection. Thus, employing GC/mass spectrometry, a specific assay allowing concurrent quantitation of the prodrug and BFA was developed involving their extraction from plasma into tert-butyl methyl ether followed by derivatization with trifluoroacetic anhydride prior to GC with mass selective detection. The lowest concentration of either BFA or prodrug quantifiable in 50 microl plasma was 0.025 microg/ml. This represents a significant improvement in sensitivity compared to previous methods, and has been shown by application to be adequate for monitoring plasma levels of either compound during the course of continuous i.v. infusion experiments. Validation and application of the method are currently in progress.
