Simple and rapid analysis of amatoxins using UPLC–MS–MS

Amatoxins, such as α-amanitin and β-amanitin, are highly toxic bicyclic octapeptides present in Amanita mushrooms. We present a simple and rapid sensitive determination of α-amanitin, β-amanitin, and phalloidin in body fluids (serum, plasma, and urine) using an ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS–MS) system. After extraction from serum, plasma, and urine samples by an Oasis HLB column, the three compounds were subjected to an UPLC–MS–MS system with an electrospray ionization interface. All three compounds were completely separated within 4.5 min. The calibration curves for serum, plasma, and urine samples showed good linearities in the range of 2–420 ng/ml, using virginiamycin B as internal standard. Their detection limits were as low as 0.5–1.5 ng/ml. The present method was validated; the coefficients of variation (CV) were: intraday for serum, plasma, and urine samples, less than 9.9, 13.2, and 11.5 %, respectively; interday for serum, plasma, and urine samples, less than 15.0, 10.4, and 15.3 %, respectively. The influence of matrix effects, especially in urine, was observed in human and rat samples; however, the difference in matrix effects between individuals were low. Therefore, each calibration curve was prepared for each biological specimen type. Furthermore, we succeeded in determining the compounds in rat urine samples, obtained 6 or 24 h after intraperitoneal administration. The present method is applicable in forensic and clinical toxicology because of its simplicity and rapidness with high sensitivity.