Thermally labile, polar toxic roridins and biologically active, isomeric baccharinoids were separated on a reversed-phase high-performance liquid chromatography column and effectively ionized under thermospray ionization conditions. The mass spectra indicated the formation of corresponding molecular ion-ammonium adducts in great abundance. Experiments designed for monitoring specific ions of these analytes at predesignated intervals were utilized for the accurate analysis of these macrocyclic trichothecenes in real, crude samples. A synthetically modified macrocyclic trichothecene, 8-ketoverrucarin A, was used as the internal standard for the detection and quantification of these compounds. Minimum detectable limits, during this first reported method for the unambiguous analysis of these structurally related macrocyclic trichothecenes, were determined to be 2-5 ng.