Novel methods for identification and quantification of the mushroom nephrotoxin orellanine - Thin-layer chromatography and electrophoresis screening of mushrooms with electron spin resonance determination of the toxin

Orellanine, (2,2'-bipyridine)-3,3',4,4'-tetrol-1,1'-dioxide, the toxin from several Cortinariaceae species, induces an acute renal failure which can be very severe or even irreversible and fatal. It is therefore important to be able to quickly and simply identify orellanine in mushroom samples with classical methods, readily available in any laboratory, such as anti-poison centers. This article reports the results of three analytical methods: classical TLC on cellulose plates in n-butanol-acetic acid-water and two original methods, electrophoresis on agarose gel and direct electron spin resonance (ESR) after enzymatic oxidation. They were applied to detect orellanine in 34 Cortinariaceae and 4 other species of toadstools. Our three sets of results are convergent. TLC (detection limit: 15 ng with fluorescence densitometry), electrophoresis (25 ng) and even ESR (5 mu g), are sensitive enough for our purpose, and a sophisticated method like HPLC (detection limit: 50 pg) is not required. As the ESR spectrum of the toxin semiquinone is highly specific, TLC or electrophoresis coupled with ESR are a convenient alternative to liquid chromatography coupled with mass spectrometry, with the same specificity, for a confirmation or with samples such as ours with high toxin contents. ESR unambiguously confirms the relatively high contents of orellanine, from 0.45% (C. henrici) to 1.1-1.4% (C. orellanus), found in five Cortinarius from the subgenus Leprocybe, section Orellani. The five species, though they are from different geographic origins, have a more or less common pattern of fluorescent compounds, among which orellinine and orelline beside orellanine. It can be useful to note that orellanine semiquinone can be easily detected by ESR directly in the fresh mushroom. The toxin is absent in the other mushrooms we tested, especially in D. cinnamomea and C. splendens, which have been claimed as toxic and suspected to contain orellanine.