Quantitative analysis of the Tricholoma ustale-derived toxin, ustalic acid, in mushroom and food samples by LC-MS/MS

Tricholoma ustale, a poisonous member of the Tricholomataceae family, causes gastrointestinal symptoms such as diarrhea and vomiting. In Japan, 86 cases (affecting a total of 347 patients) of poisoning with Tricholoma ustale have been reported between 1989 and 2010. Ustalic acid is one of the primary toxic components in Tricholoma ustale. In the present study, the quantitative analysis of the ustalic acid content in mushroom and food samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mushroom and food samples were extracted using methanol containing 0.5% formic acid and 50% aqueous methanol, respectively. Purification using SAX solid-phase extraction (SPE) was conducted prior to LC-MS/MS analysis, which was performed in the ESI negative mode using a C18 column. The method developed for the LC-MS/MS analysis of ustalic acid was extremely sensitive. The limits of quantitation calculated at a signal-to-noise ratio of 10 were 10ng/g (shiitake mushroom) and 0.40ng/g (miso soup). The accuracies of quantitation in the shiitake mushroom and miso soup samples ranged from 99.8%-105% and 98.8%-102%, respectively. This method was applied to leftover mushroom samples from a food poisoning case; here, ustalic acid was detected at 0.57, 3.7μg/g. This analytical method using LC-MS/MS could be useful in food poisoning cases involving mushrooms. This is the first report in which the ustalic acid content was determined using the leftovers of a food poisoning case.