Original article UDC: 615.918:582.28 doi:10.5633/amm.2015.0102 DETERMINATION OF MUSHROOM TOXIN ALPHA-AMANITIN IN SERUM BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY AFTER SOLID-PHASE EXTRACTION Maja Vujović 1,2 , Ivana Ilić 3 , Vesna Kilibarda 4 University of Niš, Faculty of Medicine, Department of Pharmacy, Serbia 1 Institute of Forensic Medicine, Niš, Serbia 2 University of Niš, Faculty of Medicine, Serbia 3 Military Medical Academy Belgrade, National Poison Control Centre, Serbia 4 Alpha-amanitin is a cyclic peptide which belongs to a large group of mushroom toxins known as amatoxins. Being responsible for the majority of fatal mushroom poisonings, they require rapid detection and excretion from the body fluids. In accordance with these requirements, a simple and an accurate method was developed for successful identification and quantification of alpha-amanitin in serum with electrospray liquid chromatography–mass spectrometry (LC-ESI-MS) after collision-induced dissociation. The method conforms to the established International Conference on Harmonization Q2A/Q2B 1996 guidelines on the validation of analytical methods. Linearity, precision, extraction recovery and stability test on blank serum spiked with alpha-amanitin and stored in different conditions met the acceptance criteria. The obtained calibration curve was linear over the concentration range 5-100 ng/mL with a lower limit of quantification (LOQ) of 5 ng/mL and limit of detection (LOD) of 2.5 ng/mL. The mean intra- and inter-day precision and accuracy were 6.05% and less than ±15% of nominal values, respectively. The neutral solid phase extraction with copolymer hydrophilic–lipophilic balance cartridges was found optimal for sample preparation with the mean recovery of 91.94%. The proposed method demonstrated high sensitivity and selectivity which can be useful both for clinical and forensic toxicology analysis of alpha-amanitin at low concentrations. Acta Medica Medianae 2015;54(1): 12-19. Key words: alpha-amanitin, liquid chromatography-mass spectrometry, solid phase extraction, serum Contact: Maja Vujović Bul. dr Zorana inđića 81, 18000 Niš, e-mail: majavujovic1@gmail.com Introduction Alpha-amanitin belongs to a large group of protoplasmic mushroom toxins known as ama- toxins; alpha-, beta-, gamma-, epsilon-amanitin, amanin and amanullin. Structurally, alpha-amanitin represents bicycle octapeptides with substantial toxicity effect (Figure 1) (1,2). Mush-room species like Amanita (A. phalloides, A. verna, A. virosa, A. bisporigera, A. ocreata, A. tenuifolia), also Galerina and Conocybe filaris, produce alpha-amanitin in amount sufficient to poison an adult person with liver damage and fatal outcome (LD50, p.o. humans; 0.1mg/kg) (3). Amatoxins are potent inhibitors of RNA polymerase II, essential enzyme for the synthesis of proteins, especially in liver cells (4). Several studies have shown that alpha- amanitin also exhibits prooxidant and antioxidant properties in vitro and induce disruptions of anti- oxidant enzyme defense. Free radical formation might contribute to increased levels of lipid per- oxidation and severe amatoxin hepatotoxicity (5). As the first symptoms of poisoning appear after a long lapse of time about 6-24h, alpha- amanitin is one of the most insidious poisons responsible for 80-90% of fatal mushroom poison- ing (6). Amanita intoxication has been reported to be lethal in up to 25% of the cases with mortality rate in children under ten years of age above 50 % (7). Therefore, rapid identification of alpha- amanitin in serum within 6h, when symptoms lag, and the liver and kidney damage has not occurred, is the key of successful detoxification (8). At that time, the level of amatoxins is too low for identification in urine samples utilized routine detection methods such as an immunoassay (7). Early diagnosis of poisoning via serum allows the application of appropriate therapy, avoiding expensive and invasive treatments (e.g. hemo- www.medfak.ni.ac.rs/amm 12