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Acta Medica Medianae
Vol. 54, No 1, March, 2015

UDC 61
ISSN 0365-4478(Printed version)
ISSN 1821-2794(Online)



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 Vujović1,2, Ivana Ilić3, Vesna Kilibarda4



   University of Niš, Faculty of Medicine, Department of Pharmacy, Serbia1
Institute of Forensic Medicine, Niš, Serbia2
University of Niš, Faculty of  Medicine, Serbia3
Military Medical Academy Belgrade, National Poison Control Centre, Serbia4

                          Contact: Maja Vujović
                          Bul. dr Zorana Đinđića 81, 18000 Niš, Serbia
                          e-mail: majavujovic1@gmail.com


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