Detection of α-, β-, and γ-amanitin in urine by LC-MS/MS using 15N10-α-amanitin as the internal standard

The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect alpha-, beta-, and gamma-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled N-15(10)-alpha-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the N-15(10)-alpha-amanitin internal standard, precision and accuracy of a-amanitin in pooled urine was <= 5.49% and between 100 and 106%, respectively, with a reportable range from 1-200 ng/mL. beta- and gamma-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision <= 17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0-200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled alpha-amanitin with the ability to detect and confirm human exposures to alpha-, beta-, and gamma-amanitin.