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-phas e high performance liquid chromatography (HPLC) with low wavelength UV detection or gas chromatography (GC) with electron capture detection following derivatization with heptafluorobutyrylimidazole: it was demo nstrated that breflate undergoes rapid and efficient conversion to BFA following bolus i.v. injection in mice and dogs. However, plasma conc entrations 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 mu g ml(-1)) by these methods. The sensitivity and specificity required for therapeutic drug level monito ring were achieved by GC with selected-ion mass spectrometry (MS) dete ction. Breflate, BFA and 1-eicosanol, the latter added to the sample a s an internal standard (IS), were extracted from plasma into tert-buty l methyl ether (TBME) and esterified with trifluoroacetic anhydride. M ethanol was added to the reaction mixture to effect the convenient rem oval of excess reagent as the volatile methyl ester during evaporation of the solvent. The residual material was analyzed directly upon reco nstitution by capillary GC with automated splitless injection. Electro n-ionization (70 eV) MS detection was performed by sequentially scanni ng ions at m/z 58, 202 and 325. The lowest concentration of either ana lyte quantified with acceptable reproducibility, as defined by an inte r-day R.S.D. of about 20%, was near 10 ng ml(-1) in plasma using a sam ple volume of 100 mu l. The assay has proven to be sufficiently sensit ive, specific and reproducible for the routine analysis of pharmacokin etic specimens acquired during IND (investigational new drug)-directed toxicology studies in dogs. (C) 1998 Elsevier Science B.V. All rights reserved.