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

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 (UPLC-MS/MS) method for the simultaneous determination and quantification of alpha-amanitin, beta-amanitin, gamma-amanitin, phallisacin (PSC), phallacidin (PCD), and phalloidin (POD) 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 mmx2.1 mm, 1.8 mu m) using a gradient of HPLC-grade water and methanol:0.005% formic acid. The analyte LOQs were 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 (RSD <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.