Methods to purify and determine rubratoxins.

Rubratoxin were recovered from cultures of Penicillium rubrum after the mold grew in natural substrates, a semi-synthetic medium, and a glucosemineral salts broth. Substrates that contained rubratoxins were extracted with: diethyl ether, ethyl acetate-benzene, ethanol (100%), ethanol-acetone, acetonitrile, or diethyl ether with refluxing at 45 degrees C. Extracts were screened for rubratoxins by thin-layer chromatography. Some extracts were partially purified with a column of silicic acid using acetone as the eluant. Other extracts were purified (primarily removal of pigments) using columns of silica gel plus Celite and gradient solvent elution. Most rubratoxin B (1.9 mg/g or 0.77 mg/ml) was recovered when corn, rice, or glucose-salts broth were extracted successively with diethyl ether, ethyl acetate-benzene, and diethyl ether or when samples were adjusted to pH 1.5 before refluxing with diethyl ether at 45 degrees C for 1--4h. Most rubratoxin A (0.1--0.15 mg/ml or 1.0 mg/g) was obtained from samples of corn extracted twice each with ethyl alcohol, acetone, and ethyl acetate; from glucose-mineral salts broth extracted with diethyl ether; or from yeast extract sucrose broth extracted with diethyl ether and refluxed for 4 h at 45 degrees C. Large amounts of fairly pure rubratoxin A (up to 400 mg) and rubratoxin B (greater than lg) were obtained with a combination of preparative thin-layer and column chromatography.