Forensic Science International 251 (2015) 209–213
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Simultaneous determination of mushroom toxins a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry Jana Tomkova´ *, Peter Ondra, Ivo Va´lka Department of Forensic Medicine and Medical Law, University Hospital Olomouc, Hneˇvotı´nska´ 3, 775 09 Olomouc, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2015 Received in revised form 30 March 2015 Accepted 4 April 2015 Available online 14 April 2015
This paper presents a method for the simultaneous determination of a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. The method can be used for a diagnostics of mushroom poisonings. Different SPE cartridges were tested for sample preparation, namely hydrophilic modified reversed-phase (Oasis HLB) and polymeric weak cation phase (Strata XCW). The latter gave better results and therefore it was chosen for the subsequent method optimization and partial validation. In the course of validation, limits of detection, linearity, intraday and interday precisions and recoveries were evaluated. The obtained LOD values of a-amanitin and b-amanitin were 1 ng/mL and of muscarine 0.09 ng/mL. The intraday and interday precisions of human urine spiked with a-amanitin (10 ng/mL), b-amanitin (10 ng/mL) and muscarine (1 ng/mL) ranged from 6% to 10% and from 7% to 13%, respectively. The developed method was proved to be a relevant tool for the simultaneous determination of the studied mushroom toxins in human urine after mushroom poisoning. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mushroom toxins Solid-phase extraction Liquid chromatography Mass spectrometry
1. Introduction The Amanita phalloides poisoning is rare, but may cause severe or even fatal intoxication. It is assumed that Amanita phalloides poisonings are responsible for over than 90% of the lethal cases assigned to mushrooms intoxications [1]. Amanita mushrooms contain amatoxins such as a-amanitin and b-amanitin, and phallotoxins such as phallacidin [2]. Amanitins are bicyclic octapeptides, and phallacidin is a bicyclic heptapeptide as shown in Figs. 1 and 2 [3]. Amanitins and phallotoxins are highly hepatotoxic peptides. The oral 50% lethal dose (LD50) of amanitin is approximately 0.1 mg/kg body weight – such amount may be contained in a single mushroom. Phallotoxins are not absorbed from the intestine and therefore do not play a significant role in human toxicity [4]. The Amanita phalloides mushroom poisoning has a long incubation period of 6–24 h and, in extreme cases, up to 36 h, before the sudden appearing of the gastrointestinal symptoms [5–7]. Delayed appearance of symptoms may be the problem for
* Corresponding author. Tel.: +420585639574. E-mail address: [email protected] (J. Tomkova´). http://dx.doi.org/10.1016/j.forsciint.2015.04.007 0379-0738/ß 2015 Elsevier Ireland Ltd. All rights reserved.
detectability of the Amanita phalloides mushroom poisoning. Amanitins are detectable approximately 30 h in serum and 72 h in urine after ingestion [8]. The earliest detection of the toxins in urine was reported to be 90 min after the mushroom ingestion [9]. Muscarine is the principal toxin in fungi of the genus Inocybe, Clitocybe and together with isoxazole derivatives, ibotenic acid and muscimol, is also present in the genus Amanita (Amanita pantherina, Amanita muscaria and others). It was first isolated from Amanita muscaria in 1869 (structure see Fig. 3). Muscarine was the first studied parasympathomimetic substance which causes a profound activation of the peripheral parasympathetic nervous system that may lead to convulsions and death. Muscarine poisoning is characterized by increased salivation, perspiration and lacrimation within 15–30 min after ingestion of the mushroom. With large doses, these symptoms may be followed by abdominal pain, severe nausea, diarrhea, blurred vision and labored breathing [10]. Death is rare, but it may result from cardiac or respiratory failure in severe cases [11]. The appropriate treatment is mainly symptomatic [10]. Muscarine is rapidly excreted into the urine in unmetabolised form. Mushroom poisoning is often proved by microscopic examination of spores in the stomach or intestinal content [12]. However, several analytical methods for the detection of amanitins have
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J. Tomkova´ et al. / Forensic Science International 251 (2015) 209–213
and water (all of LC–MS grade) were obtained from Sigma (St. Louis, MO, USA). The urine samples from healthy volunteers were collected and used as blank samples and as matrix for spiking with a-amantin, b-amantin, muscarine and IS. The urine samples from patients were collected during the treatment of mushrooms intoxications. 2.2. Standard solutions
Fig. 1. Chemical structures of a-amanitin (R = NH2) and b-amanitin (R = OH).
been described such as high-performance liquid chromatography [9,13], capillary electrophoresis [14], capillary electrophoresis coupled to mass spectrometry (MS) [15], liquid chromatography (LC) coupled to MS [16] or to tandem MS [1,17] or to time-of-flight MS [3] and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [2]. For determination of muscarine in biological matrices capillary electrophoresis coupled with electrospray tandem mass spectrometry [18] and liquid chromatography coupled with tandem mass [19–21] were used. In the present study a fast and sensitive method was developed for simultaneous analysis of a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction and ultra-highperformance liquid chromatography coupled with ultra-highresolution TOF mass spectrometry. a-amanitin and b-amanitin were used as diagnostic markers for Amanita phalloides poisoning. Muscarine was used as a diagnostic marker for poisonings of Amanita pantherina and Amanita muscaria. Muscarine can be used also as a diagnostic marker for poisonings of Inocybe and Clitocybe mushrooms. Phallacidin was chosen as an internal standard (IS) because it is not absorbed from the intestine. 2. Materials and methods 2.1. Chemicals and biological materials Muscarine hydrochloride, a-amanitin, b-amanitin, phallacidin, formic acid, acetic acid, sodium hydroxide, acetonitrile, methanol
A stock solution of muscarine was prepared by dissolving the standard substance in water to a concentration of 1 mg/mL. Working standard solutions of muscarine were prepared by diluting the stock standard solution with water. Stock solutions of a-amanitin, b-amanitin and phallacidin (IS) were prepared by dissolving the respective standards in methanol to yield concentrations of 1 mg/mL. Working standard solutions of a-amanitin and b-amanitin were prepared by diluting the stock standard solution with methanol. The internal standard was finally diluted with methanol to a working concentration of 10 mg/mL. 2.3. Sample preparation For the sample preparation a modified method of Merova´ et al. [19] was used. The urine sample was centrifuged at 12,100 g for 5 min and the supernatant was collected. (1) Strata X-CW procedure: Strata X-CW 30 mg cartridge (Phenomenex, Torrance, CA, USA) was preconditioned with 1 mL of methanol and 1 mL of 0.1 mol/L hydrochloric acid in water. A 1.0 mL aliquot of urine spiked with different amounts of toxins and with IS was applied to the preconditioned Strata X-CW cartridge. After washing with 1 mL of acetate buffer (pH 4.5) the cartridge was dried for 30 s and the toxins were eluted with 1 mL of 5% formic acid in methanol and then with 1 mL of methanol. The eluates were combined and evaporated to dryness with nitrogen at 30 8C. The residue was dissolved in 50 mL of mobile phase (H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid) and 5 mL of the reconstituted extract was injected in the UHPLC-UHR-TOF MS system. (2) Oasis HLB procedure: An Oasis HLB 1 cc (30 mg) cartridge (Waters, Milford, MA, USA) was preconditioned with 1 mL of methanol and 1 mL of water. A 1.0 mL aliquot of urine spiked with different amounts of toxins and with IS was applied to the preconditioned HLB cartridge. The cartridge was washed with 1 mL of 5% methanol in chloroform and analytes were eluted with 2 mL of methanol. The eluate was evaporated to dryness with nitrogen at 30 8C. The residue was dissolved in 50 mL of mobile phase (H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid) and 5 mL of the reconstituted extract was injected in the UHPLC-UHR-TOF MS system.
2.4. Instrument All analyses were carried out using a UHPLC UltiMate 3000 RSLC System (Dionex, Sunnyvale, CA, USA) connected with a UHR-TOF Maxis Impact HD (Bruker Daltonics, Billerica, MA, USA).
Fig. 2. Chemical structures of internal standard phallacidin.
Fig. 3. Chemical structures of muscarine.
J. Tomkova´ et al. / Forensic Science International 251 (2015) 209–213
Chromatographic separations were performed at 30 8C on a reverse phase analytical column Acclaim RS 120, C18 2.2 mm, 2.1 100 mm (Thermo Fisher Scientific, Waltham, MA, USA). The UHPLC UltiMate 3000 RSLC System contained binary rapid separation pumps, solvent rack with two degasser channels, an analytical in-line split loop, thermostated autosampler and a thermostated column. The chromatographic conditions were: injection volume 5 mL; flow rate 0.2 mL/min; mobile-phase solvents used (A) H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid and (B) acetonitrile/H2O 99/1 with 2 mM ammonium formate and 0.1% formic acid; gradient protocol: 1% B (0 min, 2-min hold), 30% B (5 min, 1.5-min hold), 90% B (7 min, 0.2-min hold) and 1% B (7.3 min, 0.7-min hold); total run time, 8 min. UHR-TOF Maxis Impact HD equipped with an electrosprayionisation source (ESI) operated in the positive mode. Main source settings were: capillary voltage 2500 V; nebuliser pressure 2.0 bar; drying gas flow (N2) 8 L/min; and drying temperature 200 8C. Mass spectra were collected between m/z 50 and m/z 1000. Internal mass calibration was performed at the beginning of every run, using the peaks of Na+ formate clusters. Flow rate of internal calibrant was 3 ml/mL. The mass spec resolution for all analyses was 5 ppm.
3. Results and discussion
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3.2. Optimization of UHPLC-UHR-TOF MS conditions For the separation of a-amanitin, b-amanitin and muscarine a Synergy Polar RP, 2.5 mm, 2.0 100 mm analytical column (Phenomenex, Torrance, CA, USA) was initially used but a satisfactory separation of the studied toxins was not achieved. Then we applied the reverse phase analytical column Acclaim RS 120 (see Section 2.4) which afforded adequate separation. In the course of optimization experiments the influence of flow rate, composition of mobile phase, solvent gradient and temperature of the column was tested. The final chromatographic conditions are described above. The retention time of muscarine was 2.05 min (0.05 min). The retention time of a-amanitin was 6.05 min (0.05 min). The retention time of b-amanitin was 6.08 min (0.05 min) and the retention time of the internal standard phallacidin was 6.88 min (0.05 min). In Fig. 4, extracted ion chromatograms (EIC) of human urine spiked with muscarine (1 ng/mL), a-amanitin (10 ng/mL), b-amanitin (10 ng/mL) and phallacidin (100 ng/mL) are presented. All analyses were measured in MS mode. From the molecular formula, the exact mass numbers of the protonated molecular ion and the charged molecular ion, and its isotope peaks could be easily calculated. For a-amanitin (C39H54N10O14S1 + H), theoretical calculation gave a peak at m/z 919.3614 (100%) for the protonated molecular ion, and at m/z 920.3643 (47.790%), 921.3646 (18.533%), and 922.3656 (5.184%) for its isotope peaks. For b-amanitin (C39H53N9O15S1 + H), theoretical calculation gave a peak at m/z
3.1. Optimization of the solid-phase extraction Muscarin is a low-molecular polar substance. On the contrary, amanitins are bicyclic octapeptides with high molecular weights. The aim was to achieve a single extraction procedure for muscarine, amanitins and the internal standard phallacidin. Two SPE procedures differing in type of sorbent were tested: the hydrophilic modified reversed-phase (Oasis HLB) and the polymeric weak cation phase (Strata-X-CW). The optimization was carried out with respect to elution solvents, drying times and elution volumes to achieve satisfactory recovery and to minimize working time and solvent consumption. Resulting procedures are described in Section 2.3. Both procedures were found to be comparable in respect of workload and solvent consumption, but the lower background noise and higher recoveries for all studied analytes were achieved with the procedure using Strata X-CW cartridge. Based on the results of optimization experiments the Strata X-CW procedure was selected for further validation.
Fig. 4. Extracted ion chromatograms of human urine spiked with a-amanitin (10 ng/mL), b-amanitin (10 ng/mL), muscarine (1 ng/mL), and phallacidin (IS, 100 ng/mL) obtained by UHPLC-UHR-TOF MS. Extracted ions: for a-amanitin 919.3614, for b-amanitin 920.3454, for muscarine 174.1489 and for phallacidin 847.3291.
Fig. 5. Magnified profiles of the protonated molecular ions or the charged molecular ion and their isotope peaks obtained by ultra-high-resolution TOF MS: a-amanitin: the protonated molecular ion (A), b-amanitin: the protonated molecular ion (B), muscarine: the charged molecular ion (C) and internal standard phallacidin: the protonated molecular ion (D).
J. Tomkova´ et al. / Forensic Science International 251 (2015) 209–213
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Table 1 Parameters of linearity and LOD of a-amanitin, b-amanitin and muscarine spiked into human urine, n = 5. Compound
Linear range (ng/mL)
Regression
Correlation coefficient (R2)
LOD (ng/mL)
a-amanitin b-amanitin
1–1000 1–1000 0.1–100
y = 294.27x + 2668.3 y = 268.74x + 2901 y = 6694.1x 4222.8
0.9995 0.9992 0.9994
1.0 1.0 0.1
muscarine
Table 2 Values of imprecision and recovery of spiked urine samples with different amounts of a-amanitin, b-amanitin and muscarine, n = 5. Compound
a-amanitin b-amanitin Muscarine
Concentration (ng/mL)
Precision (CV, %) Intraday
Interday
10 10 1
9.3 10.0 6.0
10.5 12.9 7.0
Recovery (%)
98 3.5 86 3.3 97 1.7
920.3454 (100%) for the protonated molecular ion, and at m/z 921.3484 (47.451%), 922.3486 (18.577%), and 923.3497 (5.219%) for its isotope peaks and for phallacidin (C37H50N8O13S1 + H), theoretical calculation gave a peak at m/z 847.3291 (100%) for the protonated molecular ion, and at m/z 848.3320 (44.812%), 849.3320 (16.961%), and 850.3331 (4.566%) for its isotope peaks. For muscarine (C9H20N1O2+), theoretical calculation gave a peak at m/z 174.1489 (100%) for the charged molecular ion, and at m/z
175.1521 (10,406%) and 176.1542 (0.899%), and for its isotope peaks. Fig. 5 shows the measured exact mass spectral data. The exact mass numbers together with their peak profiles can be used for identification of each compound by TOF MS. 3.3. Partial method validation After the optimization of sample preparation and UHPLC-UHRTOF MS conditions, the validation of the method was carried out in respect of the following analytical parameters. Limits of detection (LODs) were calculated for each analyte as a signal-to-noise ratio (S/N = 3). Linearity was tested in the range of expected concentrations. All calibration curves exhibited good linearity in the range of 0.1–100 ng/mL for muscarine and 1–1000 ng/mL for a-amanitin and b-amanitin, respectively. Intraday and interday precision for the toxins were obtained from measurements of spiked urine samples (n = 5) of a-amanitin, b-amanitin (both at 10 ng/mL) and muscarine (1 ng/mL). All validated parameters (linearity, LOD, intraday and interday precision and recovery) are summarized in Tables 1 and 2. The obtained results demonstrated that the method is applicable for analysis of proposed toxins in urine. 3.4. Analysis of patient samples
Fig. 6. UHPLC-UHR-TOF MS analysis of boy’s urine (——, full line) and negative human urine (- - -, dashed line).
Finally the urine samples from 28 patients suspected for mushroom poisoning were analyzed by means of the described SPE - UHPLC-UHR-TOF MS method. Mushroom intoxication was confirmed in two cases, namely in a 17-year old boy and an 86year old woman. In the first case Amanita muscaria poisoning was assumed because in the urine of the boy only muscarine was determined (Fig. 6). In the second case fatal Amanita phalloides poisoning was proved as in the urine of 86-year old woman a-amanitin was determined. In this case the woman died 75 h after intoxication and urine sample was taken at autopsy. The literature indicates that amanitins are detectable in urine 72 h after mushroom poisoning. In described case of fatal intoxication we determined a-amanitin in the woman’s urine 75 h after mushroom poisoning (Fig. 7). In both cases the results were confirmed by simultaneous analyses of the blank urine and the urine spiked with muscarine (1 ng/mL) and a-amanitin (10 ng/mL), respectively. 4. Conclusions The method for the simultaneous determination of a-amanitin, b-amanitin and muscarine in human urine by solid-phase
Fig. 7. UHPLC-UHR-TOF MS analysis of female’s urine (——, full line) and negative human urine (- - -, dashed line).
extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultrahigh-resolution TOF mass spectrometry was developed. All of four mushroom toxins (a-amanitin, b-amanitin, muscarine and phallacidin) were separated within 8 min. The developed method was successfully applied to the human urine spiked with studied mushroom toxins as well as to the urine samples from 28 patients suspected for mushroom poisoning from which the mushroom poisoning was proved in two cases. This method can be used for simultaneous and routine analysis of a-amanitin, b-amanitin and muscarine in human urine after intoxication by Amanita phalloides, Amanita muscaria, Amanita pantherina and other muscarine-containing mushrooms.
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References [1] M. Leite, A. Freitas, A.M. Azul, J. Barbosa, S. Costa, F. Ramos, Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples, Anal. Chim. Acta 799 (2013) 77–87. [2] K. Gonmori, K. Minakata, M. Suzuki, I. Yamagishi, H. Nozawa, K. Hasegawa, A. Wurita, K. Watanabe, O. Suzuki, MALDI-TOF mass spectrometric analysis of aamanitin, b-amanitin, and phalloidin in urine, Forensic Toxicol. 30 (2012) 179–184. [3] W.H.A. Ahmed, K. Gonmori, M. Suzuki, K. Watanabe, O. Suzuki, Simultaneous analysis of a-amanitin, b-amanitin, and phalloidin in toxic mushrooms by liquid chromatography coupled to time-of-flight mass spektrometry, Forensic Toxicol. 28 (2010) 69–76. [4] K.J. Berger, D.A. Guss, Mycotoxins revisited: Part I, J. Emerg. Med. 28 (2005) 53–62. [5] A. Mas, Mushrooms, amatoxins and the liver, J. Hepatol. 42 (2005) 166–169. [6] J. Vetter, Toxins of Amanita phalloides, Toxicon 36 (1998) 13–24. [7] M.S. Bonnet, P.W. Basson, The toxicology of Amanita phalloides, Homeopathy 91 (2002) 249–254. [8] R.C. Baselt, Disposition of toxic drugs and chemicals in man, 8th ed., Biomedical Publications, California, 2008. [9] C. Defendenti, E. Bonacina, M. Mauroni, L. Gelosa, Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine, Forensic Sci. Int. 92 (1998) 59–68. [10] D. Michelot, L.M. Melendez-Howell, Amanita muscaria: chemistry, biology, toxicology, and ethnomycology, Mycol. Res. 107 (2003) 131–146. [11] J.H. Halpern, Hallucinogens and dissociative agents naturally growing in the United States, Pharmacol. Ther. 102 (2004) 131–138. [12] J. Strˇı´brny´, M. Sokol, B. Merova´, P. Ondra, GC/MS determination of ibotenic acid and muscimol in the urine of patients intoxicated with Amanita pantherina, Int. J. Leg. Med. 126 (2012) 519–524.
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[13] G. Caccialanza, C. Gandini, R. Ponci, Direct, simultaneous determination of alphaamanitin, beta-amanitin and phalloidine by high-performance liquid chromatography, J. Pharm. Biomed. Anal. 3 (1985) 179–185. [14] V.A. Robinson-Fuentes, J.L. Jaime-Sa´nchez, L. Garcı´a-Aguilar, M. Go´mez-Peralta, M.S. Va´zquez-Garciduen˜as, G. Va´zquez-Marrufo, Determination of alpha- and beta-amanitin in clinical urine samples by capillary zone electrophoresis, J. Pharm. Biomed. Anal. 47 (2008) 913–917. [15] J. Rittgen, M. Pu¨tz, U. Pyell, Identification of toxic oligopeptides in Amanita fungi employing capillary electrophoresis-electrospray ionization-mass spectrometry with positive and negative ion detection, Electrophoresis 29 (2008) 2094–2100. [16] H.H. Maurer, C.J. Schmitt, A.A. Weber, T. Kraemer, Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction, J. Chromatogr. B Biomed. Sci. Appl. 748 (2000) 125–135. [17] A.G. Helfer, M.R. Meyer, J.A. Michely, H.H. Maurer, Direct analysis of the mushroom poisons a- and b-amanitin in human urine using a novel on-line turbulent flow chromatography mode coupled to liquid chromatography-high resolutionmass spectrometry/mass spectrometry, J. Chromatogr. A 1325 (2014) 92–98. [18] P. Ginterova´, B. Sokolova´, P. Ondra, J. Znaleziona, J. Petr, J. Sˇevcˇı´k, V. Maier, Determination of mushroom toxins ibotenic acid, muscimol and muscarine by capillary electrophoresis coupled with electrospray tandem mass spektrometry, Talanta 125 (2014) 242–247. [19] B. Merova´, M. Stanˇkova´, J. Strˇı´brny´, P. Ondra, LC–MS – an objective diagnostic method of Amanita intoxications, Chem. Listy 106 (2012) 831–835. [20] B. Merova´, P. Ondra, M. Stanˇkova´, M. Soural, J. Strˇı´brny´, L. Hebka´, K. Lemr, Determination of muscarine in human urine by electrospray liquid chromatographic-mass spectrometric, J. Chromatogr. B 879 (2011) 2549–2553. [21] B. Merova´, P. Ondra, M. Stanˇkova´, I. Va´lka, Isolation and identification of the Amanita muscaria and Amanita pantherina toxins in human urine, Neuroendocrinol. Lett. 29 (2008) 744–748.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Simultaneous determination of mushroom toxins a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry Jana Tomkova´ *, Peter Ondra, Ivo Va´lka Department of Forensic Medicine and Medical Law, University Hospital Olomouc, Hneˇvotı´nska´ 3, 775 09 Olomouc, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2015 Received in revised form 30 March 2015 Accepted 4 April 2015 Available online 14 April 2015
This paper presents a method for the simultaneous determination of a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. The method can be used for a diagnostics of mushroom poisonings. Different SPE cartridges were tested for sample preparation, namely hydrophilic modified reversed-phase (Oasis HLB) and polymeric weak cation phase (Strata XCW). The latter gave better results and therefore it was chosen for the subsequent method optimization and partial validation. In the course of validation, limits of detection, linearity, intraday and interday precisions and recoveries were evaluated. The obtained LOD values of a-amanitin and b-amanitin were 1 ng/mL and of muscarine 0.09 ng/mL. The intraday and interday precisions of human urine spiked with a-amanitin (10 ng/mL), b-amanitin (10 ng/mL) and muscarine (1 ng/mL) ranged from 6% to 10% and from 7% to 13%, respectively. The developed method was proved to be a relevant tool for the simultaneous determination of the studied mushroom toxins in human urine after mushroom poisoning. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mushroom toxins Solid-phase extraction Liquid chromatography Mass spectrometry
1. Introduction The Amanita phalloides poisoning is rare, but may cause severe or even fatal intoxication. It is assumed that Amanita phalloides poisonings are responsible for over than 90% of the lethal cases assigned to mushrooms intoxications [1]. Amanita mushrooms contain amatoxins such as a-amanitin and b-amanitin, and phallotoxins such as phallacidin [2]. Amanitins are bicyclic octapeptides, and phallacidin is a bicyclic heptapeptide as shown in Figs. 1 and 2 [3]. Amanitins and phallotoxins are highly hepatotoxic peptides. The oral 50% lethal dose (LD50) of amanitin is approximately 0.1 mg/kg body weight – such amount may be contained in a single mushroom. Phallotoxins are not absorbed from the intestine and therefore do not play a significant role in human toxicity [4]. The Amanita phalloides mushroom poisoning has a long incubation period of 6–24 h and, in extreme cases, up to 36 h, before the sudden appearing of the gastrointestinal symptoms [5–7]. Delayed appearance of symptoms may be the problem for
* Corresponding author. Tel.: +420585639574. E-mail address: [email protected] (J. Tomkova´). http://dx.doi.org/10.1016/j.forsciint.2015.04.007 0379-0738/ß 2015 Elsevier Ireland Ltd. All rights reserved.
detectability of the Amanita phalloides mushroom poisoning. Amanitins are detectable approximately 30 h in serum and 72 h in urine after ingestion [8]. The earliest detection of the toxins in urine was reported to be 90 min after the mushroom ingestion [9]. Muscarine is the principal toxin in fungi of the genus Inocybe, Clitocybe and together with isoxazole derivatives, ibotenic acid and muscimol, is also present in the genus Amanita (Amanita pantherina, Amanita muscaria and others). It was first isolated from Amanita muscaria in 1869 (structure see Fig. 3). Muscarine was the first studied parasympathomimetic substance which causes a profound activation of the peripheral parasympathetic nervous system that may lead to convulsions and death. Muscarine poisoning is characterized by increased salivation, perspiration and lacrimation within 15–30 min after ingestion of the mushroom. With large doses, these symptoms may be followed by abdominal pain, severe nausea, diarrhea, blurred vision and labored breathing [10]. Death is rare, but it may result from cardiac or respiratory failure in severe cases [11]. The appropriate treatment is mainly symptomatic [10]. Muscarine is rapidly excreted into the urine in unmetabolised form. Mushroom poisoning is often proved by microscopic examination of spores in the stomach or intestinal content [12]. However, several analytical methods for the detection of amanitins have
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J. Tomkova´ et al. / Forensic Science International 251 (2015) 209–213
and water (all of LC–MS grade) were obtained from Sigma (St. Louis, MO, USA). The urine samples from healthy volunteers were collected and used as blank samples and as matrix for spiking with a-amantin, b-amantin, muscarine and IS. The urine samples from patients were collected during the treatment of mushrooms intoxications. 2.2. Standard solutions
Fig. 1. Chemical structures of a-amanitin (R = NH2) and b-amanitin (R = OH).
been described such as high-performance liquid chromatography [9,13], capillary electrophoresis [14], capillary electrophoresis coupled to mass spectrometry (MS) [15], liquid chromatography (LC) coupled to MS [16] or to tandem MS [1,17] or to time-of-flight MS [3] and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [2]. For determination of muscarine in biological matrices capillary electrophoresis coupled with electrospray tandem mass spectrometry [18] and liquid chromatography coupled with tandem mass [19–21] were used. In the present study a fast and sensitive method was developed for simultaneous analysis of a-amanitin, b-amanitin and muscarine in human urine by solid-phase extraction and ultra-highperformance liquid chromatography coupled with ultra-highresolution TOF mass spectrometry. a-amanitin and b-amanitin were used as diagnostic markers for Amanita phalloides poisoning. Muscarine was used as a diagnostic marker for poisonings of Amanita pantherina and Amanita muscaria. Muscarine can be used also as a diagnostic marker for poisonings of Inocybe and Clitocybe mushrooms. Phallacidin was chosen as an internal standard (IS) because it is not absorbed from the intestine. 2. Materials and methods 2.1. Chemicals and biological materials Muscarine hydrochloride, a-amanitin, b-amanitin, phallacidin, formic acid, acetic acid, sodium hydroxide, acetonitrile, methanol
A stock solution of muscarine was prepared by dissolving the standard substance in water to a concentration of 1 mg/mL. Working standard solutions of muscarine were prepared by diluting the stock standard solution with water. Stock solutions of a-amanitin, b-amanitin and phallacidin (IS) were prepared by dissolving the respective standards in methanol to yield concentrations of 1 mg/mL. Working standard solutions of a-amanitin and b-amanitin were prepared by diluting the stock standard solution with methanol. The internal standard was finally diluted with methanol to a working concentration of 10 mg/mL. 2.3. Sample preparation For the sample preparation a modified method of Merova´ et al. [19] was used. The urine sample was centrifuged at 12,100 g for 5 min and the supernatant was collected. (1) Strata X-CW procedure: Strata X-CW 30 mg cartridge (Phenomenex, Torrance, CA, USA) was preconditioned with 1 mL of methanol and 1 mL of 0.1 mol/L hydrochloric acid in water. A 1.0 mL aliquot of urine spiked with different amounts of toxins and with IS was applied to the preconditioned Strata X-CW cartridge. After washing with 1 mL of acetate buffer (pH 4.5) the cartridge was dried for 30 s and the toxins were eluted with 1 mL of 5% formic acid in methanol and then with 1 mL of methanol. The eluates were combined and evaporated to dryness with nitrogen at 30 8C. The residue was dissolved in 50 mL of mobile phase (H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid) and 5 mL of the reconstituted extract was injected in the UHPLC-UHR-TOF MS system. (2) Oasis HLB procedure: An Oasis HLB 1 cc (30 mg) cartridge (Waters, Milford, MA, USA) was preconditioned with 1 mL of methanol and 1 mL of water. A 1.0 mL aliquot of urine spiked with different amounts of toxins and with IS was applied to the preconditioned HLB cartridge. The cartridge was washed with 1 mL of 5% methanol in chloroform and analytes were eluted with 2 mL of methanol. The eluate was evaporated to dryness with nitrogen at 30 8C. The residue was dissolved in 50 mL of mobile phase (H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid) and 5 mL of the reconstituted extract was injected in the UHPLC-UHR-TOF MS system.
2.4. Instrument All analyses were carried out using a UHPLC UltiMate 3000 RSLC System (Dionex, Sunnyvale, CA, USA) connected with a UHR-TOF Maxis Impact HD (Bruker Daltonics, Billerica, MA, USA).
Fig. 2. Chemical structures of internal standard phallacidin.
Fig. 3. Chemical structures of muscarine.
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Chromatographic separations were performed at 30 8C on a reverse phase analytical column Acclaim RS 120, C18 2.2 mm, 2.1 100 mm (Thermo Fisher Scientific, Waltham, MA, USA). The UHPLC UltiMate 3000 RSLC System contained binary rapid separation pumps, solvent rack with two degasser channels, an analytical in-line split loop, thermostated autosampler and a thermostated column. The chromatographic conditions were: injection volume 5 mL; flow rate 0.2 mL/min; mobile-phase solvents used (A) H2O/acetonitrile 99/1 with 2 mM ammonium formate and 0.1% formic acid and (B) acetonitrile/H2O 99/1 with 2 mM ammonium formate and 0.1% formic acid; gradient protocol: 1% B (0 min, 2-min hold), 30% B (5 min, 1.5-min hold), 90% B (7 min, 0.2-min hold) and 1% B (7.3 min, 0.7-min hold); total run time, 8 min. UHR-TOF Maxis Impact HD equipped with an electrosprayionisation source (ESI) operated in the positive mode. Main source settings were: capillary voltage 2500 V; nebuliser pressure 2.0 bar; drying gas flow (N2) 8 L/min; and drying temperature 200 8C. Mass spectra were collected between m/z 50 and m/z 1000. Internal mass calibration was performed at the beginning of every run, using the peaks of Na+ formate clusters. Flow rate of internal calibrant was 3 ml/mL. The mass spec resolution for all analyses was 5 ppm.
3. Results and discussion
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3.2. Optimization of UHPLC-UHR-TOF MS conditions For the separation of a-amanitin, b-amanitin and muscarine a Synergy Polar RP, 2.5 mm, 2.0 100 mm analytical column (Phenomenex, Torrance, CA, USA) was initially used but a satisfactory separation of the studied toxins was not achieved. Then we applied the reverse phase analytical column Acclaim RS 120 (see Section 2.4) which afforded adequate separation. In the course of optimization experiments the influence of flow rate, composition of mobile phase, solvent gradient and temperature of the column was tested. The final chromatographic conditions are described above. The retention time of muscarine was 2.05 min (0.05 min). The retention time of a-amanitin was 6.05 min (0.05 min). The retention time of b-amanitin was 6.08 min (0.05 min) and the retention time of the internal standard phallacidin was 6.88 min (0.05 min). In Fig. 4, extracted ion chromatograms (EIC) of human urine spiked with muscarine (1 ng/mL), a-amanitin (10 ng/mL), b-amanitin (10 ng/mL) and phallacidin (100 ng/mL) are presented. All analyses were measured in MS mode. From the molecular formula, the exact mass numbers of the protonated molecular ion and the charged molecular ion, and its isotope peaks could be easily calculated. For a-amanitin (C39H54N10O14S1 + H), theoretical calculation gave a peak at m/z 919.3614 (100%) for the protonated molecular ion, and at m/z 920.3643 (47.790%), 921.3646 (18.533%), and 922.3656 (5.184%) for its isotope peaks. For b-amanitin (C39H53N9O15S1 + H), theoretical calculation gave a peak at m/z
3.1. Optimization of the solid-phase extraction Muscarin is a low-molecular polar substance. On the contrary, amanitins are bicyclic octapeptides with high molecular weights. The aim was to achieve a single extraction procedure for muscarine, amanitins and the internal standard phallacidin. Two SPE procedures differing in type of sorbent were tested: the hydrophilic modified reversed-phase (Oasis HLB) and the polymeric weak cation phase (Strata-X-CW). The optimization was carried out with respect to elution solvents, drying times and elution volumes to achieve satisfactory recovery and to minimize working time and solvent consumption. Resulting procedures are described in Section 2.3. Both procedures were found to be comparable in respect of workload and solvent consumption, but the lower background noise and higher recoveries for all studied analytes were achieved with the procedure using Strata X-CW cartridge. Based on the results of optimization experiments the Strata X-CW procedure was selected for further validation.
Fig. 4. Extracted ion chromatograms of human urine spiked with a-amanitin (10 ng/mL), b-amanitin (10 ng/mL), muscarine (1 ng/mL), and phallacidin (IS, 100 ng/mL) obtained by UHPLC-UHR-TOF MS. Extracted ions: for a-amanitin 919.3614, for b-amanitin 920.3454, for muscarine 174.1489 and for phallacidin 847.3291.
Fig. 5. Magnified profiles of the protonated molecular ions or the charged molecular ion and their isotope peaks obtained by ultra-high-resolution TOF MS: a-amanitin: the protonated molecular ion (A), b-amanitin: the protonated molecular ion (B), muscarine: the charged molecular ion (C) and internal standard phallacidin: the protonated molecular ion (D).
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Table 1 Parameters of linearity and LOD of a-amanitin, b-amanitin and muscarine spiked into human urine, n = 5. Compound
Linear range (ng/mL)
Regression
Correlation coefficient (R2)
LOD (ng/mL)
a-amanitin b-amanitin
1–1000 1–1000 0.1–100
y = 294.27x + 2668.3 y = 268.74x + 2901 y = 6694.1x 4222.8
0.9995 0.9992 0.9994
1.0 1.0 0.1
muscarine
Table 2 Values of imprecision and recovery of spiked urine samples with different amounts of a-amanitin, b-amanitin and muscarine, n = 5. Compound
a-amanitin b-amanitin Muscarine
Concentration (ng/mL)
Precision (CV, %) Intraday
Interday
10 10 1
9.3 10.0 6.0
10.5 12.9 7.0
Recovery (%)
98 3.5 86 3.3 97 1.7
920.3454 (100%) for the protonated molecular ion, and at m/z 921.3484 (47.451%), 922.3486 (18.577%), and 923.3497 (5.219%) for its isotope peaks and for phallacidin (C37H50N8O13S1 + H), theoretical calculation gave a peak at m/z 847.3291 (100%) for the protonated molecular ion, and at m/z 848.3320 (44.812%), 849.3320 (16.961%), and 850.3331 (4.566%) for its isotope peaks. For muscarine (C9H20N1O2+), theoretical calculation gave a peak at m/z 174.1489 (100%) for the charged molecular ion, and at m/z
175.1521 (10,406%) and 176.1542 (0.899%), and for its isotope peaks. Fig. 5 shows the measured exact mass spectral data. The exact mass numbers together with their peak profiles can be used for identification of each compound by TOF MS. 3.3. Partial method validation After the optimization of sample preparation and UHPLC-UHRTOF MS conditions, the validation of the method was carried out in respect of the following analytical parameters. Limits of detection (LODs) were calculated for each analyte as a signal-to-noise ratio (S/N = 3). Linearity was tested in the range of expected concentrations. All calibration curves exhibited good linearity in the range of 0.1–100 ng/mL for muscarine and 1–1000 ng/mL for a-amanitin and b-amanitin, respectively. Intraday and interday precision for the toxins were obtained from measurements of spiked urine samples (n = 5) of a-amanitin, b-amanitin (both at 10 ng/mL) and muscarine (1 ng/mL). All validated parameters (linearity, LOD, intraday and interday precision and recovery) are summarized in Tables 1 and 2. The obtained results demonstrated that the method is applicable for analysis of proposed toxins in urine. 3.4. Analysis of patient samples
Fig. 6. UHPLC-UHR-TOF MS analysis of boy’s urine (——, full line) and negative human urine (- - -, dashed line).
Finally the urine samples from 28 patients suspected for mushroom poisoning were analyzed by means of the described SPE - UHPLC-UHR-TOF MS method. Mushroom intoxication was confirmed in two cases, namely in a 17-year old boy and an 86year old woman. In the first case Amanita muscaria poisoning was assumed because in the urine of the boy only muscarine was determined (Fig. 6). In the second case fatal Amanita phalloides poisoning was proved as in the urine of 86-year old woman a-amanitin was determined. In this case the woman died 75 h after intoxication and urine sample was taken at autopsy. The literature indicates that amanitins are detectable in urine 72 h after mushroom poisoning. In described case of fatal intoxication we determined a-amanitin in the woman’s urine 75 h after mushroom poisoning (Fig. 7). In both cases the results were confirmed by simultaneous analyses of the blank urine and the urine spiked with muscarine (1 ng/mL) and a-amanitin (10 ng/mL), respectively. 4. Conclusions The method for the simultaneous determination of a-amanitin, b-amanitin and muscarine in human urine by solid-phase
Fig. 7. UHPLC-UHR-TOF MS analysis of female’s urine (——, full line) and negative human urine (- - -, dashed line).
extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultrahigh-resolution TOF mass spectrometry was developed. All of four mushroom toxins (a-amanitin, b-amanitin, muscarine and phallacidin) were separated within 8 min. The developed method was successfully applied to the human urine spiked with studied mushroom toxins as well as to the urine samples from 28 patients suspected for mushroom poisoning from which the mushroom poisoning was proved in two cases. This method can be used for simultaneous and routine analysis of a-amanitin, b-amanitin and muscarine in human urine after intoxication by Amanita phalloides, Amanita muscaria, Amanita pantherina and other muscarine-containing mushrooms.
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