Simple and rapid detection of three amatoxins and three phallotoxins in human body fluids by UPLC–MS-MS and its application in 15 poisoning cases

Abstract

Abstract Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of foodborne sickness and poisoning-related fatalities in China. This study introduces and validates a simple, rapid and cost-effective ultra-performance liquid chromatography–mass spectrometry method for the simultaneous determination and quantification of α-amanitin, β-amanitin, γ-amanitin, phallisacin, phallacidin and phalloidin in human blood and urine. Quick therapeutic decision-making is supported by a 9 min chromatographic separation performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) using a gradient of high-performance liquid chromatography (HPLC)-grade water and methanol:0.005% formic acid. The analyte limit of quantification was 1–3 ng/mL in blood and 0.5–2 ng/mL in urine. Calibrations curves, prepared by spiking drug-free blood and urine, demonstrated acceptable linearity with mean correlation coefficients (r) greater than 0.99 for all phallotoxins and amatoxins. Acceptable intraday and interday precision (relative standard deviation <15%) and accuracy (bias, −4.8% to 13.0% for blood and—9.0% to 14.7% for urine) were achieved. The validated method was successfully applied to analyze 9 blood samples and 2 urine samples testing positive for amatoxins and/or phallotoxins. Amatoxins and/or phallotoxins were identified in each whole blood sample at a range of 1.12–5.63 ng/mL and in two urine samples from 1.01–9.27 ng/mL. The method has the benefits of simple sample preparation (protein precipitation) and wide analyte coverage, making it suitable for emergency quantitative surveillance toxicological analysis in clinics and forensic poisoning practice.