Journal of Analytical Toxicology 2014;38:335–340 doi:10.1093/jat/bku035 Advance Access publication April 28, 2014 Article

Amatoxins (a- and b-Amanitin) and () Analyses in Urines Using High-Resolution Accurate Mass LC–MS Technology

Thomas Gicquel1, Sylvie Lepage1, Manon Fradin1, Olivier Tribut2,Be´ne´dicte Duretz3 and Isabelle Morel1*

1Laboratoire de Toxicologie Biologique et Me´dico-le´gale, CHU Pontchaillou, F-35033 Rennes, France, 2UF Biomarqueurs, CHU Pontchaillou, F-35033 Rennes, France and 3Thermo Fisher, Courtaboeuf, France

*Author to whom correspondence should be addressed. Email: [email protected]

Mycotoxin intoxications can result from the consumption of amatox- The toxicity results from an inhibition of RNA synthesis by the ins like a- and b-amanitin or of phallotoxin, present in several toxic , leading to cell necrosis (3), especially in liver, Downloaded from https://academic.oup.com/jat/article-abstract/38/6/335/2797989 by guest on 19 April 2020 like phalloides. To identify and quantify amatox- and intestinal cells (4). Indeed, phalloid syndrome often leads to ins and phallotoidin in biological matrixes, we developed a method hepatic failure, which is fatal in 10–40% of intoxication cases and using liquid chromatography coupled with an ultra-high-resolution often requires hepatic transplantation (5, 6). The first symptoms and accurate mass instrument (liquid chromatography–high-resolution- appear within 24 h, after a period of latency before hepatic and mass spectrometry, LC–HR-MS), Q ExactiveTM (Thermo Fisher). The renal failure (7). Patients usually arrive at the hospital 12 h after method includes a simple solid-phase extraction of urine samples ingestion of the mushrooms, since symptoms appear after a spiked with flurazepam as internal standard (IS), using Bond Elut corresponding lag time. At that time, most of the amatoxins Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was have already been eliminated from the plasma (6–8). Detection performed on a C18 Accucore column (100 3 2.1 mm, 2.6 mm) of in plasma can only be made for a 36-h period after using a gradient of 10 mM ammonium acetate buffer containing ingestion (6), whereas their detection in urines can begin as soon 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic as 90 min until almost 4 days after ingestion (6, 9). Therefore, acid. Separation of analytes was obtained in 7 min, with respective urines represent the most appropriate sample for detection of retention times for a-amanitin, b-amanitin, phalloidin and IS of 1.9, mycotoxins in poisoning. Moreover, since clinical 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC–HR-MS sys- manifestations are not pathognomonic, it is important to identify tem was performed by extracting the exact mass value of each pro- and quantify the mycotoxins which are involved. Specific and fast tonated species using a 5-p.p.m. mass window, which was 919.3614, detection of amatoxins in body fluids is necessary for an early 920.3455, 789.3257 and 388.1586 for a-amanitin, b-amanitin, phalloi- diagnosis of intoxication. To quantify mycotoxins in biological din and IS, respectively. Calibration curves were obtained by spiking fluid and to be able to give a therapy as quickly as possible, tox- drug-free urine at 1–100 ng/mL. Mean correlation coefficients, r2, icologists must adapt and improve their analytical techniques to were above 0.99 for each amatoxins and phalloidin. According to cur- make them faster and more reliable. Various analytical methods rently accepted validation procedures, the method was tested for have been already developed to identify and quantify amatoxins selectivity, calibration, accuracy, matrix effect, precision and recov- in urines, such as capillary electrophoresis (10, 11)orliquid ery. Authentic urine samples from 43 patients suffering from a sus- chromatography–mass spectrometry (LC–MS) (8, 9, 12, 13). pected intoxication with mushrooms were analyzed by LC–HR-MS, Other nonseparative methods could be used such as radioimmu- and the results were compared with ELISA competitive immunoas- noassay (14, 15) or ELISA competitive immunoassay (2, 16). say. The LC–HR-MS presented large benefits over immunoassay of To our knowledge, only one recent publication using high- being specific, faster and more sensitive, making it suitable for resolution-mass spectrometry/mass spectrometry (HR-MS–MS) daily emergency toxicological analysis. wasreportedtoidentifyamatoxins(a-andb-amanitin) in urines, with a full validated method restricted to a-amanitin only (17). No method has already been developed and validated for simultane- Introduction ous quantification of low levels of a-amanitin, b-amanitin and phal- Every year, ingestion of toxic mushrooms causes many cases of loidin in a limited volume of authentic urines from patients. illnesses and can lead to death in the absence of medical assis- For the first time, we describe here a full validated method using liquid chromatography coupled with an ultra-HR and accurate tance. The phalloid syndrome is estimated to be responsible for TM 95% of fatal cases of mushroom poisonings throughout the mass instrument (Q Exactive ) (LC–HR-MS) for simultaneous a b world. Intoxications can result from the consumption of quantification of -amanitin, -amanitin and phalloidin in a limited like amatoxins (a-amanitin, b-amanitin, g-amanitin, 1-amanitin, volume of urine samples. Urines from 43 patients collected from and ) or (phalloidin and phalloin), declared cases to poison centers all around France and supposed present in several toxic mushrooms such as Amanita, Lepiota or to suffer from a intoxication with mushrooms were analyzed using Galerina species. These mushrooms are responsible for the most this validated LC–HR-MS method, and results were compared severe cases of with a very high mortality rate with those obtained using an ELISA competitive immunoassay. (1). Phalloidin, a-amanitin and b-amanitin represent the major tox- ins in . These toxins are cyclopeptides belong- ing to two groups of toxicologically different compounds: the Experimental amatoxins, which are lethal within 2–8 days and highly toxic Chemicals and reagents (LD50 ¼ 0.2–0.6 mg/kg) and the phallotoxins, which are less a-Amanitin (C39H54N10O14S), b-amanitin (C39H53N9O15S), phal- toxic than the amatoxins (LD50 ¼ 2–3 mg/kg) (1, 2). loidin (C35H48N8O11S), formic acid, ammonium acetate and

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] flurazepam, as the internal standard (IS), were purchased from 3,000 g, the supernatants were loaded on Bond Elut Agilent Sigma-Aldrich (St. Louis, MO, USA). , acetonitrile and C18 (200 mg, 3 mL) columns previously conditioned with water were obtained from Fisher Scientific UK (Loughborough, 2 mL of methanol, 2 mL of distilled water and 2 mL of the dilution Leicestershire, UK). Ammonia was purchased from Probalo buffer. The columns were then washed with 4 mL of LC–MS (Paris, France). All chemicals, reagents and solvents were of water after sample loading. The analytes were then eluted with LC–MS quality grade. 3 mL of methanol with 2% of ammonia. The supernatants were evaporated to dryness at 508C under a stream of nitrogen. Residues were dissolved in 200 mL of mobile phase and trans- Instrumentation ferred for LC–HR-MS analysis. Analyses were performed on a Thermo Scientific Q ExactiveTM (San Jose, USA) mass spectrometer including an Accela pump (Thermo Scientific, San Jose, CA, USA). An heated electrospray ionisation-II Selectivity (HESI-II) ion source was used for the ionization of target com- Six different sources of blank urine were extracted and analyzed, pounds. Data acquisition, peak integration and calibration were and no interferences were found at lower limit of quantification Downloaded from https://academic.oup.com/jat/article-abstract/38/6/335/2797989 by guest on 19 April 2020 performed using the Xcaliburw 2.1 software (Thermo Scientific). (LLOQ) levels. Eight blank samplesfortifiedwithISwereana- lyzed to check for the absence of interfering signals.

LC conditions LC separation was performed using a gradient on a C18 Accucore Calibration and linearity column (100 mm 2.1, 2.6 mm). The mobile phases used were Seven-point calibration curves were obtained by fortifying drug- 10 mM ammonium acetate buffer containing 0.1% (v/v) formic free human urine with working solution of a-amanitin, b-amanitin acid (solvent A) and acetonitrile with 0.1% (v/v) formic acid (sol- and phalloidin at final concentrations of 1, 2, 5, 10, 20, 50 and vent B). The mobile phase was delivered at a flow rate of 100 ng/mL. After extraction, calibration curves were run each 400 mL/min using the following stepwise gradient elution program: day in duplicate. Standard curves corresponded to peak area ratios initial conditions of 90 : 10 (A : B) run from 89 : 11 (A : B) at 2 min, of each analytes to IS using weighted linear least-squares regres- run from 40 : 60 (A : B) at 3 min, maintained from 3 to 4.5 min, run sion (1/x2) and coefficient of determination (r2) were calculated. from 20 : 80 (A : B) at 4.5 min and conditions 90 : 10 (A : B) main- tained to 7 min for equilibration. All prepared samples were kept Precision and accuracy (trueness) at 158C in the autosampler until injection of 10 mL into the LC– HR-MS system (partial loop) in a thermostated column at 308C. Precision and accuracy were determined using 500 mLofhuman urine samples supplemented with known concentrations (1, 5 and 50 ng/mL) of a-amanitin, b-amanitin and phalloidin in the MS conditions same batch. The repeatability (intraday precision) was evaluated The MS conditions were as follows: HESI in the positive mode, by analyzing six different samples in the same day. For intermediate capillary temperature: 3008C; spray voltage: 3,500 V; sheath and precision (interday precision), six different day analyses were as- auxiliary gas (nitrogen) flow rate: 40 psi and 10 (arbitrary units), sessed at the same concentration. The calculated values were respectively. Data are acquired in Targeted SIM (single ion mon- based on a daily calibration curve. Precision was calculated by itoring) mode. Tuning parameters were optimized by direct infu- using the coefficient of variation, CV % ¼ (SD/M) (100); sion of individual analytes (a-amanitin, b-amanitin, phalloidin where M is the mean of the experimentally determined concentra- and IS) at the concentration of 1 mg/L in the mobile phase tions and SD the standard deviation of M. Accuracy (trueness) was into the ionization probe at a flow rate of 5 mL/min in electro- calculated using the bias; bias ¼ jE 2 Tj (absolute value), where E spray ionisation positive mode. Resolution was set to 70,000 was the experimentally determined concentration and T the theo- full width half maximum at 200 a.m.u. and AGC Target was 5e5. retical concentration. The assay acceptance criterion for each con- Maximum injection time was 100 ms and isolation width 4.0 m/z. centration was +15% deviation of the nominal concentration, The measured accurate m/z values of the protonated species except for the LLOQ where a deviation of +20% was accepted. were 919.3614, 920.3455, 789.3257 and 388.1586 for a-amanitin, b-amanitin, phalloidin and IS, respectively. Quantitation was per- Recovery, process efficiency and matrix effect formed by extracting the exact mass of each of the protonated species using a 5-p.p.m. extraction window. Recovery (RE), matrix effects (MEs) and process efficiency (PE) were performed at two concentrations (2 and 20 ng/mL) in anal- ogy to the simplified approach described by Matuszewski et al. Standard solutions (18). Briefly, RE was calculated by comparing average peak Stock solutions of a-amanitin, b-amanitin, phalloidin and IS were areas of drug-free human urine fortified by the same concentra- prepared in methanol (1.0 g/L) and then stored at 48C. tions, before or after the extraction procedure. PE was determined by the ratio of average peak areas from ex- tracted drug-free human urine previously fortified by the com- Sample preparation pounds tested and from samples prepared at the same nominal Urine samples (500 mL) were supplemented with 200 pg of IS concentrations in water (neats). and extracted by solid-phase extraction (SPE). After dilution of ME was calculated as follows: (100 mean peak area of forti- the urine samples with 1 mL of the dilution buffer (formic acid fied drug 2 free human urine after extraction/mean peak area of 1%; pH 2), sonication 10 min and centrifugation 10 min at neats) 2 100.

336 Gicquel et al. Patients analysis detection of analytes in a limited volume of 500 mL of urine sam- Fifty urine samples, collected from 43 patients (25 men/18 ple, requiring lower volumes of solvents and reagents. Stability of women, including 3 children) suspected to have consumed amanitins in urine samples have already been investigated and toxic mushroom between January 2011 and August 2013, were showed that these compounds were stable during sample collec- analyzed by LC–HR-MS, and the results compared with ELISA tion, storage, handling and analysis (8). kits. Samples have been store at 2208C until analysis.

Separation and specificity/selectivity Amanitin quantification by the ELISA competitive Parameters have been chosen to provide satisfactory separation immunoassay and peak shapes for all analytes and IS in only 7 min. Retention Toxins, a-amanitin and g-amanitin were measured in urine sam- times of a-amanitin, b-amanitin, phalloidin and IS were, respec- ples using the Amanitin ELISA kit according to the manufactur- tively, 1.9, 1.7 and 3.5 min (Figure 1). However, the total run er’s procedure (Bu¨hlmann, Switzerland). time of the method was set to 7 min in order to eliminate all in- terferences and re-equilibrate the column for the next injection. Downloaded from https://academic.oup.com/jat/article-abstract/38/6/335/2797989 by guest on 19 April 2020 Statistical analysis Blank urine samples from different sources did not show any in- terfering peaks influencing the analytes or the IS signal. Our run The results are expressed as means + standard deviation (SD). A is shorter than the other reported LC–MS methods (8, 9, 12, 13, D’Agostino–Pearson normality test was performed to evaluate 17, 21, 22). Furthermore, the amanitins, a and b, which present normality and a Spearman’s rank correlation test was used to es- structural homologies were chromatographically separated timate the strength of the relationship between two sets of data. under the conditions. A P-value of ,5% was considered statistically significant.

Results and discussion Method validation During method development, different procedures have been Linearity evaluated to optimize sample extraction, ME and chromato- graphic and detection parameters. Standard curves for a-amanitin, b-amanitin and phalloidin were linear over the range of 1–100 ng/mL. A calibration curve was es- tablished for each analyte using linear regression analysis of the Sample preparation ratio of analyte peak area/IS peak area versus analyte concentra- Sample preparation and chromatographic conditions of extrac- tion with a weighting factor of 1/x. tion have been carefully optimized for simple, rapid and practical The linearity was verified with a lack of fit test and the coeffi- quantitative analysis, avoiding most of MEs. Most of the published cient of determination, r2,was.0.99 for a-amanitin, b-amanitin methods involved solid–liquid extraction of samples, using large and phalloidin. Typical equations of calibration curves were as volumes of urine in order to reach detectable levels (8, 9, 12, follows: a-amanitin: y ¼ 0.0021 þ 0.0392x and r2 ¼ 0.9950; 19, 20). In our study, an SPE without derivatization allowed b-amanitin: y ¼ 0.0060 þ 0.0238x and r2 ¼ 0.9907; phalloidin:

Figure 1. Chromatograms of protonated a-amanitin, b-amanitin and phalloidin obtained by accurate mass extraction using a 5-p.p.m. mass window. Amatoxins and phalloidin were extracted from urine spiked at the concentration of 1 ng/mL.

Amatoxin Analysis by Accurate Mass Technology 337 y ¼ 20.0416 þ 0.171056x and r2 ¼ 0.9912. Lower limit of dete- Urine sample analysis and comparison with ction were 0.25, 0.5 and 0.25 ng/mL and LLOQ were 0.5, 0.75 the ELISA method and 0.5 ng/mL, respectively, for a-amanitin, b-amanitin and Authentic urine samples from patients have been analyzed using phalloidin. our validated LC–HR-MS method and compared with an ELISA method. Globally in authentic urines, a-amanitin was the most Precision and accuracy abundant and the most frequently detected using Precision and trueness were determined using urine samples for- the separative method of LC–HR-MS. tified with known concentrations of a-amanitin, b-amanitin and The concentrations obtained by these two methods were in phalloidin concentrations. Each sample was analyzed and the good correlation (Figure 2). We observed a linear regression statisti- intra- and interday means, SD, CV % and bias were calculated. cally significant different from zero (P , 0.0001). A D’Agostino– The intraday precision and trueness for urines spiked with Pearson normality test was performed and showed that the 1ng/mL were 5.9, 14.7 and 14.3% and 20.1, 0.1 and 0.1 ng/mL, values were not normally distributed. Hence, a Spearman’s rank respectively, for a -amanitin, b-amanitin and phalloidin (Table I). correlation test showed a consistent and significant correlation Downloaded from https://academic.oup.com/jat/article-abstract/38/6/335/2797989 by guest on 19 April 2020 The interday precision and trueness for urines spiked with between LC–HR-MS and ELISA results, with a coefficient of 1ng/mL were 14.6, 14.5 and14.8% and 0.1, 0.1 and 0.1 ng/mL, re- 0.9303 and a P-value two-tailed was ,0.0001. spectively, for a-amanitin, b-amanitin and phalloidin (Table II). In addition, using the ELISA method, the calibration curve ob- The precision of IS was 10.2% for intraday and 9.3% for interday tained at 450 nm was linear only in the range of concentrations precision, respectively. between 2 and 30 ng/mL. For a larger range of concentrations Critical problems in the analysis of mushroom toxins are the ab- such as the one used for LC–HR-MS (from 1 to 100 ng/mL), sence of readily available deuterated IS. After tested current IS such the sigmoid curve led to imprecision in extreme concentrations, as microcystin RR and antibiotics, which did not give satisfaction in decreasing correlation parameters calculated between these two termofRE,wehavefinallychosenflurazepam(MW¼ 387.8782) as methods. the IS. This compound did not completely fullfill requirements for The ELISA method showed two false-negative results and four the choice of an IS, but it appeared to be suitable for obtaining the false-positive results when compared with LC–HR-MS (Table IV). full validation of our method. This could be partly explained by differences in the detection of amanitins since the ELISA method detected both a-and Recovery, process efficiency and matrix effect g-amanitins simultaneously (2), whereas LC–HR-MS can detect RE ranged from 88.4 to 93.4% and PE from 93.2 to 108.5% a-amanitin, b-amanitin and phalloidin separately. (Table III). ME was evaluated for all analytes, and toxins showed ion enhancement not higher than 20%.

Table III Table I RE, PE and ME for quantification in spiked urine (data are mean of n ¼ 6 analyses) Intraday precision and accuracy for a-amanitin, b-amanitin and phalloidin quantification in spiked urine (data are mean + SD, n ¼ 6) Compounds RE % PE % ME %

Compounds C (ng/mL) Mean + SD (ng/mL) Precision (CV %) Accuracy (ng/mL) 2ng/mL 20 ng/mL 2 ng/mL 20 ng/mL 2 ng/mL 20 ng/mL a -Amanitin 1 0.9 + 0.1 5.9 0.1 a-Amanitin 93.4 91.6 99.8 92.7 6.8 1.3 5 5.2 + 0.5 9.5 0.2 b-Amanitin 88.4 90.6 93.2 103.3 5.4 14.0 50 49.9 + 1.9 3.8 0.1 Phalloidin 90.5 91.1 108.5 91.5 20.0 0.5 b-Amanitin 1 1.0 + 0.1 14.7 0.1 5 5.2 + 0.6 11.2 0.2 50 49.9 + 1.9 3.8 0.1 Phalloidin 1 1.1 + 0.2 14.3 0.1 5 5.1 + 0.4 7.3 0.1 50 49.9 + 3.4 6.8 0.1

SD, standard deviation; CV, coefficient of variation.

Table II Interday precision and accuracy for a-amanitin, b-amanitin and phalloidin quantification in spiked urine (data are mean + SD, n ¼ 6)

Compounds C (ng/mL) Mean + SD (ng/mL) Precision (CV %) Accuracy (ng/mL) a-Amanitin 1 1.1 + 0.2 14.6 0.1 5 4.9 + 0.5 10.4 0.1 50 48.7 + 2.7 5.6 1.3 b-Amanitin 1 1.1 + 0.2 14.5 0.1 5 5.5 + 0.7 12.8 0.5 50 53.2 + 4.1 7.8 3.2 Phalloidin 1 1.1 + 0.2 14.8 0.1 5 4.9 + 0.6 12.4 0.1 50 46.9 + 5.1 10.9 3.1 Figure 2. Comparison of urine concentrations obtained by LC–HR-MS analysis versus SD, standard deviation; CV, coefficient of variation. ELISA competitive immunoassay.

338 Gicquel et al. Table IV interferences with urine matrix, drugs or their metabolites? Comparison of results in authentic urine samples analyzed by ELISA kits and by LC–HR-MS Toxichem Krimtech, 68, 68–71. 3. Wieland, T., Go¨tzendo¨rfer, C., Zanotti, G., Vaisus, A.C. (1981) The ef- Urine samples Correlation fect of the chemical nature of the side chains of amatoxins in the in- ELISA LC–HR-MS hibition of eukaryotic RNA polymerase B. European Journal of Biochemistry, 117, 161–164. FN 2 0 4. Wieland, T., Faulstich, H. (1991) Fifty years of amanitin. Experientia, FP 4 0 47, 1186–1193. N2426 P21255. Floersheim, G.L., Weber, O., Tschumi, P., Ulbrich, M. (1982) Clinical death-cap (Amanita phalloides) poisoning: prognostic factors and FN, false negative; FP, false positive; N, true negative; P, true positive. therapeutic measures. Analysis of 205 cases. Schweiz Med Wochenschr, 112, 1164–1177. 6. Jaeger, A., Jehl, F., Flesch, F., Sauder, P., Kopferschmitt, J. (1993) Moreover, the false-positive rate with ELISA could also be as a Kinetics of amatoxins in human poisoning: therapeutic implications. Journal of Toxicology Clinical Toxicology, 31, 63–80.

result of other metabolites of these mushroom toxins, such as Downloaded from https://academic.oup.com/jat/article-abstract/38/6/335/2797989 by guest on 19 April 2020 the glucuronides, sulfates or hydroxylated products, which 7. Barbato, M.P. (1993) Poisoning from accidental ingestion of mush- were present in the urine and captured by the antibody. rooms. The Medical Journal of Australia, 158, 842–847. 8. Maurer, H.H., Schmitt, C.J., Weber, A.A., Kraemer, T. (2000) Validated Finally, some urine samples have been analyzed with the electrospray liquid chromatographic–mass spectrometric assay for ELISA method in 2011 and with LC–HR-MS 2 years later, sug- the determination of the mushroom toxins a-andb-amanitin in gesting a possible degradation of mycotoxins during a pro- urine after immunoaffinity extraction. Journal of Chromatography longed storage at 2208C. However, Maurer et al. (8)and B, 748, 125–135. Robinson-Fuentes et al. (11) have reported that a-and 9. Defendenti, C., Bonacina, E., Mauroni, M., Gelosa, L. (1998) Validation b-amanitin were stable in urine for a period of at least 6 months of a high performance liquid chromatographic method for alpha am- anitin determination in urine. Forensic Science International, 92, at 48C. Nevertheless, no information is available for a longer pe- 59–68. riod of storage. 10. Bru¨eggemann, O., Meder, M., Freitag, R. (1996) Analysis of amato- The discrepancy could also be explained by differences in xins alpha-amanitin and beta-amanitin in toadstool extracts LLOQ between the two methods, since the ELISA test was char- and body fluids by capillary zone electrophoresis with photo- acterized for an LLOQ of 1.5 ng/mL (2) and we determined an diode array detection. Journal of Chromatography A, 744, LLOQ of ,1ng/mL by LC–HR-MS, leading to false-negative re- 167–176. sults with the ELISA method. It is, however, interesting to note 11. 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340 Gicquel et al.