A specific, sensitive analytical method was developed for the quantitative determination of brefeldin A (NSC 89671) in mouse plasma. The assay utilizes capillary gas-liquid chromatography with electron-capture detection. Commercially available 1-eicosanol was found to be a suitable internal standard. The drug and internal standard were extracted from 50 pl of plasma with diethyl ether, the solvent removed by evaporation and the residue treated with a perfluoroacylating reagent to yield the electron-capturing derivatives. Aliquots of the derivative mixtures were then introduced onto a fused silica capillary column, which is wall-coated with cross-linked methyl silicone gum. The effluent from the gas chromatographic column as monitored using a 63Ni electron capture detector. Standard curves were prepared by plotting peak area ratios of brefeldin A to the internal standard against the brefeldin A concentration. The best fit straight line was determined by least squares regression using a weighting factor of reciprocal peak area squared to calculate the slope, y-intercept and correlation coefficient. Brefeldin A concentrations in unknown samples were calculated using the results of the regression analysis. The concentration range of the present assay extends from 0.1 micrograms/ML to 2.2 micrograms/ML, and its precision and accuracy over that range are 10% or better. The lower limit of quantitation is 0.1 micrograms/mL, while the lower limit of detection is about 0.025 micrograms/mL. The present capillary gas chromatographic assay is appropriate for the preliminary investigation of brefeldin A pharmacokinetics in mice.
