Simple and sensitive determination of α- and β-amanitin by liquid chromatography–quadrupole time-of-flight mass spectrometry

Abstract

Amatoxins, including aand b-amanitins, are highly toxic cyclic octapeptide compounds belonging to the Amanita species; these toxins exert gastroenteric symptoms in the early stage, and lead to hepatic failure and renal damage by inhibiting RNA polymerase activity in hepatocytes [1–3]. The Amanita species are considered to be responsible for more than 90 % of the lethal cases of mushroom toxin poisoning [4, 5]. It is thus important to detect amatoxins with high sensitivity. Recently, liquid chromatography (LC)–mass spectrometry (MS) [6–13] and tandem mass spectrometry (MS/MS) [14–18] or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOFMS) [19] have been increasingly applied to detect and identify these compounds from body fluids or mushroom specimens. In this Letter, we present a new method for detecting and identifying aand b-amanitin using a novel LC–quadrupole time-of-flight mass spectrometer (Q-TOFMS) with high sensitivity and specificity. Amanitins were purchased from Sigma (St. Louis, MO, USA); other chemicals used were of the highest purity commercially available. Urine samples were collected from a healthy volunteer after obtaining informed consent. Analyses by Q-TOFMS were performed on an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA) coupled to a Shimadzu Prominence XR LC system (Shimadzu, Kyoto, Japan). For LC separation, a Scherzo SM-C18 column (2 9 100 mm, particle size 3 lm, Imtakt, Kyoto, Japan) was used; the column temperature was kept at 40 C and the flow rate was 0.5 ml/ min. The gradient program was started at 90 % mobile phase A (5 mM ammonium formate in distilled water) and 10 % mobile phase B (methanol) for 1 min; it was changed linearly to 5 % A and 95 % B over 4 min and maintained with a 2-min hold. The MS conditions were as follows: ionization mode, electrospray ionization (ESI) positive mode; turbo gas temperature, 500 C; spray voltage, 5,000 V. In the MS mode, ions were scanned in the range from m/z 850 to 1,000; the declustering potential (DP) and collision energy (CE) were 80 V and 10 V, respectively. In the MS/MS mode, four conditions for production ion monitoring were settled as follows: precursor ions were 919.4 for a-amanitin and 920.4 for b-amanitin, and product ions were scanned in the range of m/z 200–1,000 (DP, 80 V; CE, 50 V); and precursor ions were 919.4 for a-amanitin and 920.4 for b-amanitin, and product ions were scanned in the range of m/z 900–1,600 (DP, 80 V; CE, 10 V). Measurements in MS and MS/MS modes were employed alternatively, for 100 ms in each condition; the total cycle time was 550 ms. Authentic amanitins were dissolved in methanol or added to purified urine samples to assess the sensitivity of the instrument and evaluate matrix effects. In the samples diluted in methanol, the concentrations of both amanitins were in the range of 0.001–5 ng/ll. In urine samples, 200 ll of urine was mixed with 100 ll of acetonitrile and centrifuged at 5,000 rpm for 10 min. The supernatant was mixed with 700 ll of distilled water and A. Ishii (&) M. Kusano K. Zaitsu Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: akishii@med.nagoya-u.ac.jp