Analysis of α- and β-amanitin in Human Plasma at Subnanogram per Milliliter Levels by Reversed Phase Ultra-High Performance Liquid Chromatography Coupled to Orbitrap Mass Spectrometry

Abstract

Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and β-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37–2890 pg/mL, and β-amanitin was found in seven plasma samples ranging from <20–7520 pg/mL. A LC-HRMS/MS method for the quantitation of amatoxins in human blood plasma at subnanogram per milliliter levels was developed, validated, and used for the analysis of plasma samples. The method provides a valuable alternative to urine analysis, allowing thorough patient treatment but also further study the toxicokinetics of amatoxins.

Further development of a liquid chromatography–high‐resolution mass spectrometry/mass spectrometry‐based strategy for analyzing eight biomarkers in human urine indicating toxic mushroom or Ricinus communis ingestions

“…So far, several analytical methods have been published that allow detection of biomarkers of one mushroom intoxication syndrome or Ricinus seed intoxication in human urine or plasma. 29,[36][37][38][39][40][41][42][43][44] Tomkov a et al developed a method for a simultaneous determination of αand β-amanitin and muscarine in urine by LC-high-resolution (HR)-time-of-flight (TOF) MS. 45 Recently, we presented a comprehensive and validated analytical solution to identify a total of eight biomarkers of castor bean intoxication, phalloides syndrome, pantherine-muscaria syndrome, muscarine syndrome, psilocybin syndrome, and bufotenine intoxication in urine within one single run. 18 The samples were prepared by SPE and urine precipitation performed in parallel and the use of a four-eluent system for normal-phase chromatography were necessary to realize the aims of selectivity and earmarked sensitivity for all analytes.…”

“…In clinical toxicology, time‐saving comprehensive methods are favorable, because they allow fast decision making and a rapid start of a specific therapy. So far, several analytical methods have been published that allow detection of biomarkers of one mushroom intoxication syndrome or Ricinus seed intoxication in human urine or plasma 29,36–44 . Tomková et al developed a method for a simultaneous determination of α‐ and β‐amanitin and muscarine in urine by LC‐high‐resolution (HR)‐time‐of‐flight (TOF) MS 45 .…”

Selected Instrumental Techniques Applied in Food and Feed: Quality, Safety and Adulteration Analysis

“…Naturally occurring toxins include mycotoxins [ 312 , 313 ], marine biotoxins [ 314 , 315 ], cyanogenic glycosides [ 316 ], and toxins from poisonous mushrooms (e.g., γ-amanitin, ustalic acid) [ 317 , 318 ]. In the latter example, toxins are usually measured mostly in biological samples such as plasma, serum, and urine as measure of exposure [ 319 , 320 ].…”

A label-free electrochemical immunosensor based on decorated cellulose nanofibrous membrane for point-of-care diagnosis of amanitin poisoning via human urine

“…In patients with suspected amatoxin poisoning, treatment should be initiated immediately based on clinical suspicion, even before laboratory results are available. 7–9…”

“…7 Laboratory-based analysis is typically required, using techniques such as HPLC, mass spectrometry, or ELISA to detect the presence of amatoxins in blood, urine, or mushroom extracts. 7,8 The diagnosis of amatoxin poisoning is usually based on a combination of clinical symptoms, history of mushroom ingestion, and laboratory results. In patients with suspected amatoxin poisoning, treatment should be initiated immediately based on clinical suspicion, even before laboratory results are available.…”