Analysis of brefeldin A in plasma by gas chromatography with electron capture detection

The National Cancer Institute (NCI) is pursuing preclinical development of Brefeldin A (BFA), a macrolide isolated from Penicillium brefeldianum, as an antitumor agent. BFA exhibits a unique spectrum of activity against the human tumor cell line panel that composes the NCI's recently established in vitro antitumor screen. A specific method to assay the compound in biological fluids has been developed in which BFA and 1-eicosanol, added as the internal standard, are extracted from plasma specimens with diethyl ether. The residue afforded by evaporation of the organic solvent is dried in vacuo and derivatized with heptafluorobutyrylimidazole prior to analysis by capillary gas chromatography with electron capture detection. Derivatization of both secondary hydroxyl groups of BFA is rapid and quantitative, as confirmed by mass spectrometry. The lowest concentration of BFA quantified with acceptable reproducibility in 50 microliters of plasma is 0.1 microgram/ml, for which a 5.6% coefficient of variation has been determined from six standard curves assayed on separate days. Detector response exhibits a positive deviation from linearity for samples with BFA concentrations greater than 2.5 micrograms/ml. The assay is shown by application to be suitable for preliminary investigations of BFA plasma pharmacokinetics in mice. These studies reveal that BFA is subject to rapid elimination from the mouse, with plasma levels declining in an apparent biphasic manner from an initial concentration of 33 to 0.2 micrograms/ml at 60 min after intravenous treatment with a 26.3 mg/kg dose.