High-pressure liquid chromatography of the mycotoxins, rubratoxins A and B, and its application to the analysis of urine and plasma for rubratoxin B.

The rubratoxins are toxic metabolites produced by Penicillium rubrum and P. purpurogenum on food and feedstuffs. Rubratoxin B is hepatotoxic, mutagenic and teratogenic. Rubratoxins A and B were resolved as sharp peaks in the order A-B by reversed-phase high-pressure liquid chromatography on a small-particle (10 micrometer) column in 3 min by an acetonitrile-water-ethyl acetate elution solvent (11:9.9:3), with detection by ultraviolet absorbance at 254 nm. The relationship between peak height and quantity injected was linear over a range of 0.25-5 microgram for rubratoxin A and 0.05-5 microgram for rubratoxin B. Retention time and peak height and peak area were highly reproducible for both toxins. Detection was very sensitive, allowing detection of 3-5 ng rubratoxin B, and 15-20 ng rubratoxin A. Quantitative recovery of rubratoxin B from spiked urine samples was obtained over a range of 5-40 microgram/ml, with maximum recovery from urine samples adjusted to pH 2 before extraction. Good recovery of rubratoxin B was also obtained from spiked plasma samples subjected to treatment with 3 N hydrochloric acid followed by extraction with ethyl acetate.