Direct analysis of the mushroom poisons α- and β-amanitin in human urine using a novel on-line turbulent flow chromatography mode coupled to liquid chromatography–high resolution-mass spectrometry/mass spectrometry

• Introduction of a new TurboFlow (PQEM) mode for bioanalysis.
• Simple, fast, and robust method for detection of α- and β-amanitin in human urine.
• Application to analysis of authentic urine samples containing amatoxins.
• Description of pseudo HR-MS3 studies as powerful tool in compound identification.

Poisonings with Amanita phalloides toxins require fast diagnosis in order to avoid expensive and unnecessary therapies. Initial clinical assessment in combination with urinary amanitin analysis is necessary for a definite diagnosis. Therefore, a simple, fast, and robust method was developed for reliable detection of α- and β-amanitin as well as for fully validated quantification of α-amanitin in human urine. After simple dilution and centrifugation of the urine sample, a fast on-line extraction using a Transcend TLX-II system based on turbulent flow chromatography (TurboFlow) was established. A new TurboFlow mode was introduced, the pseudo quick elute mode (PQEM), which had more options for method optimization than the generic quick elute mode (QEM). It allowed running several modes in one valve arrangement. The PQEM showed better practicability in routine and emergency analysis than the previously used methods. After extraction, the fast 15 min LC–high resolution (HR)-MS/MS analysis allowed reliable identification of α- and β-amanitin based on fragments identified using so-called HR pseudo MS3 experiments. According to international recommendations, the requirements for full validation including the parameters selectivity, calibration, accuracy, precision, recovery, matrix effects, and stability were fulfilled for α-amanitin. The method was successfully applied to the analysis of authentic urine samples containing amatoxins. In conclusion, this method allowed the determination of amatoxins using the novel PQEM in a faster, robust, and more reliable way than existing methods, making it suitable for daily routine and especially emergency toxicological analysis.