Liquid Chromatography Mass Spectrometry Urinalysis & Mushroom Poisoning

Mushroom poisoning is a serious health concern that can result in acute illness and even death. Although the definitive number of people affected by mycotoxin-related illness is unknown, the nature of this opportunistic pathogen renders it a serious health concern on an international scale.¹ The ingestion of Amanita phalloides, colloquially known as the “death cap” mushroom, is exceptionally toxic, producing more than 90% of the fatalities attributed to mushroom poisoning.² The major mycotoxins active in this mushroom are cyclopeptide amatoxins α-amanitin and β-amanitin and the cyclopeptide phallotoxin phalloidin. Specific, rapid urinalysis for the identification of the mycotoxins responsible is imperative for prompt treatment.

For this reason, Gicquel et al. composed a liquid chromatography–ultra high-resolution mass spectrometry (LC-HRMS) method for simultaneous quantification of α-amanitin, β-amanitin and phalloidin in urine samples.³ To do this, the team used samples collected from 43 patients with reported mushroom intoxication and compared the results to enzyme-linked immunosorbent assay (ELISA) for validation. Their method relied upon solid-phase extraction with a flurazepam internal standard on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer, using an Accela pump and Heated Electrospray Ionization-II probe (Thermo Scientific). The team used Xcalibur software (version 2.1, Thermo Scientific) for data acquisition and processing. The researchers set experimental parameters that allowed for accurate separation and peaks for all analytes and the internal standard within seven minutes. Retention times were 1.9, 1.7 and 3.5 minutes, respectively, for α-amanitin (m/z 919.3614), β-amanitin (m/z 920.3455) and phalloidin (m/z 789.3257).

Using spiked urine, the researchers established calibration curves over a concentration range of 1 to 100 ng/mL, verified linearity, and determined correlation coefficients above 0.99. They report the following lower limits of detection and quantification, respectively, for the “heated electrospray ionization-II ion probe”toxins: α-amanitin, 0.25 and 0.5 ng/mL; β-amanitin, 0.5 and 0.75 ng/mL; and phalloidin, 0.25 and 0.5 ng/mL. The team reports the protocol is highly reproducible and presents the following intraday and interday values (% RSD), respectively: α-amanitin, 5.9% and 14.6%; β-amanitin, 14.7% and 14.5%; and phalloidin, 14.3% and 14.8%. The ranges for recovery and process efficiency were 88.4 to 93.4% and 93.2 to 108.5%, respectively. The value for matrix effects never exceeded 20% for ion enhancement.

When the team compared the LC-HRMS data with ELISA for validation purposes, they found good correlation, and a Spearman’s rank revealed a 0.9303 coefficient and <0.0001 two-tailed p-value. Overall, Gicquel et al. verified that their method meets criteria for selectivity, accuracy, calibration, matrix effect, precision and recovery. Moreover, the method offers increased sensitivity and rapidity as compared to ELISA for validation purposes, rendering it ideal for emergency urinalysis. Given the association between A. phalloides and high levels of toxicity, these findings may point to alternative or complementary protocols for acute clinical applications.

References

1. Bennett, J.W., and Klich, M. (2003) “Mycotoxins,” Clinical Microbiology Reviews, 16(3) (pp. 497–516).

2. Roberts, D.M., et al. (2013) “Amanita phalloides poisoning and treatment with silibinin in the Australian Capital Territory and New South Wales,” Medical Journal of Australia, 198(1) (pp. 43–7).

3. Gicquel, T., et al. (2014, July) “Amatoxins (α- and β-Amanitin) and Phallotoxin (Phalloidin) Analyses in Urines Using High-Resolution Accurate Mass LC–MS Technology,” Journal of Analytical Toxicology, 38(6) (pp. 335–40), doi: 10.1093/jat/bku035.

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.