Quantification of Filipin and Antimycin A

Filipin and antimycin A production was assessed after growth at 30°C in YEME medium without sucrose. To assay filipin in culture broths, 1 volume of culture was extracted with 1 volume of methanol and was further diluted with methanol to bring the absorbance at 338 nm into the range of 0.1 to 0.4 units. Solutions of pure filipin III (Sigma) were used as controls. The identity of filipin was confirmed by analysis of its UV-visible absorption spectrum (absorption peaks at 356, 338, 320, and 311 nm). Quantitative determination of filipin was performed as previously described (26 (link)), using a Mediterranea Sea C₁₈ column (Teknokroma) (4.6 by 150 mm; particle size, 3 mm). For antimycin A production assessment, 1 volume of culture was extracted with 2 volumes of ethyl acetate and was dried by rotary evaporation. The pellet was then resuspended in methanol prior to HPLC analysis. The same chromatographic method was used for estimation of antimycin A production at 318 nm. Pure antimycin A (Sigma) was used as the standard.