Analysis of brefeldin A and the prodrug breflate in plasma by gas chromatography with mass selective detection

Breflate is a water soluble prodrug developed to facilitate parenteral administration of the investigational antineoplastic agent brefeldin A (BFA). Previously, using analytical methods based upon reversed-phase high performance liquid chromatography (HPLC) with low wavelength UV detection or gas chromatography (GC) with electron capture detection following derivatization with heptafluorobutyrylimidazole, it was demonstrated that breflate undergoes rapid and efficient conversion to BFA following bolus i.v. injection in mice and dogs. However, plasma concentrations of the drug and prodrug achieved during the administration of nontoxic doses of breflate to beagle dogs as a 72 h continuous i.v. infusion were undetectable (<0.1 μg ml^?1) by these methods. The sensitivity and specificity required for therapeutic drug level monitoring were achieved by GC with selected-ion mass spectrometry (MS) detection. Breflate, BFA and 1-eicosanol, the latter added to the sample as an internal standard (IS), were extracted from plasma into tert-butyl methyl ether (TBME) and esterified with trifluoroacetic anhydride. Methanol was added to the reaction mixture to effect the convenient removal of excess reagent as the volatile methyl ester during evaporation of the solvent. The residual material was analyzed directly upon reconstitution by capillary GC with automated splitless injection. Electron-ionization (70 eV) MS detection was performed by sequentially scanning ions at m/z 58, 202 and 325. The lowest concentration of either analyte quantified with acceptable reproducibility, as defined by an inter-day R.S.D. of about 20%, was near 10 ng ml^?1 in plasma using a sample volume of 100 μl. The assay has proven to be sufficiently sensitive, specific and reproducible for the routine analysis of pharmacokinetic specimens acquired during IND (investigational new drug)-directed toxicology studies in dogs.