Article pubs.acs.org/JAFC
Detection and Quantitative Analysis of the Non-cytotoxic alloTenuazonic Acid in Tomato Products by Stable Isotope Dilution HPLC-MS/MS Sebastian Hickert,†,‡ Isabel Krug,† Benedikt Cramer,† and Hans-Ulrich Humpf*,†,‡ †
Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany NRW Graduate School of Chemistry, Wilhelm-Klemm-Strasse 10, 48149 Münster, Germany
‡
S Supporting Information *
ABSTRACT: Tenuazonic acid (1) is a mycotoxin produced mainly by fungi of the genus Alternaria. It occurs in a variety of agricultural products. allo-Tenuazonic acid (2) is an isomer of 1 that is not chromatographically separated from 1 in most analytical methods. Therefore, both isomers are quantitated as a sum parameter. In this study a QuEChERS (quick, easy, cheap, effective, rugged and safe) based stable isotope dilution HPLC-MS/MS method including the chromatographic separation of both isomers was developed and applied to 20 tomato products from the German market. All products showed contamination with both toxins. 1 was found in a range from 5.3 ± 0.1 to 550 ± 15 μg/kg (average = 120 μg/kg) and 2 in a range from 1.5 ± 0.4− to 270 ± 0.8 μg/kg (average = 58 μg/kg). 2 represents 7.0−44% of the sum of both isomers (average = 29%). This is the first reported occurrence of 2 in food samples. To evaluate and compare the cytotoxicities of 1 and 2, both compounds were isolated from a synthetic racemic mixture. 1 showed moderate cytotoxic effects on HT-29 cells starting at 100 μM, whereas 2 exhibited no activity. 2 was not produced in liquid cultures of Alternaria alternata in yeast extract sucrose (YES) medium, but could be detected in small amounts in tomato puree inoculated with the fungus. KEYWORDS: mycotoxin, alternaria, mass spectrometry, stable isotope dilution assay, liquid chromatography, cytotoxicity
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INTRODUCTION Tenuazonic acid ((5S,8S)-3-acetyl-5-sec-butyltetramic acid, 1) is a toxic secondary metabolite formed mainly by Alternaria fungi and was first isolated by Rosett et al.1 and structurally assigned by Stickings et al.2 The toxicity of Alternaria metabolites has been reviewed.3,4 1 shows acute toxic effects in rodents (oral LD50 for mice, 81−186 mg/kg bw; and for rats, 168−180 mg/ kg bw)5 and chicken embryos (LD50 0.55 mg/egg).6 One mode of action in mice is the inhibition of protein synthesis.7 A shortterm animal trial (33 days) on monkeys led to vomiting, bloody diarrhea, and hemorrhagic lesions in the intestine after treatment with 89.6 mg/kg bw per day.8 In the first publication describing 1, an isomerization product after lengthy standing at room temperature was described.1 Joshi et al.9 reported the presence of an analogue of 1 in culture extracts from Alternaria tenuis on rice, which was not further characterized. The presence of allo-tenuazonic acid ((5R,8S)-3-acetyl-5-sec-butyltetramic acid, 2) in fungal culture extracts (Alternaria brassicicola, Alternaria raphani, and Phoma sorghina) has been shown.10,11 1 epimerizes to a mixture of 1 and 2 when treated with bases1,2,10 and under acidic conditions.12 Figure 1 shows the structures of 1 and 2. 1 and mixtures of 1 and 2 are phytotoxic, and the phytotoxicity is not influenced by the proportion of 1 and 2.13 The phytotoxic effects of 1 and 2 can be explained by the inhibitory effects of 1 on photosynthesis by blocking the electron flow.14 There are no further toxicological data regarding 2 found in the literature. Some synthetic analogues (D-tenuazonic acid, ((5S,8R)-3-acetyl-5-secbutyltetramic acid) and D-allo-tenuazonic acid ((5R,8R)-3acetyl-5-sec-butyltetramic acid)) with the unnatural D-config© 2015 American Chemical Society
Figure 1. Structures of 1 ((5S,8S)-3-acetyl-5-sec-butyltetramic acid) and 2 ((5R,8S)-3-acetyl-5-sec-butyltetramic acid).
uration of the isoleucine moiety of 1 have been tested for their antitumor, cytotoxic, and antibacterial activities,15 but this study did not cover L-allo-tenuazonic acid (with the naturally occurring L-isoleucine). Antitumor activities for 1, as well as cytotoxicity on Eagle’s KB cell carcinoma were shown. The analogues showed strong differences in their activity against carcinoma cells but not in their activity against Bacillus megaterium.15 This emphasizes the importance of the configuration for toxicological activities of 1 analogues. There are numerous methods for the quantification of 1 published. 1 occurs, for example, in tomato products,16,17 apple products,18 and grain products.19 None of these methods can distinguish between 1 and 2. There are some HPLC applications for the (partial) separation of 1 and 2 published, which use ion pair reagents, Received: Revised: Accepted: Published: 10879
October 2, 2015 December 2, 2015 December 3, 2015 December 3, 2015 DOI: 10.1021/acs.jafc.5b04812 J. Agric. Food Chem. 2015, 63, 10879−10884
Article
Journal of Agricultural and Food Chemistry ligand exchange, or anion exchange chromatography20,21 and high carbon load columns.10,12 Some of these methods are not transferrable to HPLC-MS due to the use of nonvolatile additives. A promising approach to achieve better chromatographic behavior on RP-HPLC columns for 1 is the derivatization with 2,4-dinitrophenylhydrazine (DNPH).17,22 Despite the improved retention, no separation of 1 and 2 was reported. None of the mentioned methods detected 2 in food samples. The scope of this study was the preparative isolation of 1 and 2 for cytotoxicity testing and the development of a HPLC-MS/ MS stable isotope dilution assay for the simultaneous detection of both isomers in tomato products. Furthermore, the ability of four strains of Alternaria alternata to produce 2 in liquid medium and in tomato puree was investigated. To study whether 2 is formed during food processing, naturally contaminated tomato purees were concentrated at elevated temperature to simulate the production of tomato paste.
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Separation of 1 and 2 for Cytotoxicity Assays. The racemic mixture of 1 and 2 from our previous synthesis16 was fractionated by preparative HPLC using a Supelco Ascentis RP-Amide column (2500 × 10 mm, 5 μm particle size, Sigma-Aldrich, Steinheim, Germany). An amount of 50.4 mg of the mixture was dissolved in 25 mL of MeOH/ H2O/FA (20:79:1, v/v/v). A HPLC system with a PU-2087 highpressure gradient pump coupled to a UV-2075 UV detector (Jasco Labor and Datentechnik, Gross-Umstadt, Germany) was used. The separation occurred isocratically with MeOH/H2O/FA (52:47:1, v/v/ v) and was monitored by UV detection at 277 nm; 2.4 mL was injected per run. 1 and 2 eluted as one broad peak from 18.6 to 22.0 min. This broad peak was fractioned in three fractions; the first fraction was collected from 18.6 to 19.6 min, fraction 2 from 19.6 to 21.0 min, and fraction 3 from 21.0 to 22.0 min. The fractions of 10 HPLC runs were pooled, and fractions 1 and 3 were separately fractionated for a second time. Again, three fractions as mentioned were taken. All three pooled fractions were evaporated to dryness using a rotary evaporator, dissolved in water, and freeze-dried. The isomeric purity was determined by 1H NMR on a DPX-400 spectrometer (Bruker Biospin, Rheinstetten, Germany) and by HPLC-MS/MS. First fraction (2): 1H NMR (400 MHz, CDCl3) δ 3.86 (s, H-5, 5R,8S), 2.45 (s, H-7, 5R,8S), 2.09−1.94 (m, H-8), 1.54−1.25 (m, H9), 0.96 (d, J = 7.5 Hz, H-11, 5R,8S), 0.79 (t, J = 6.7 Hz, H-10, 5R,8S). Second fraction (mixture of 1 and 2): 1H NMR (400 MHz, CDCl3) δ 3.78 (s, H-5, 5S,8S), 3.86 (s, H-5, 5R,8S), 2.50 (s, H-7, 5S,8S), 2.45 (s, H-7, 5R,8S), 2.05−1.91 (m, H-8), 1.48−1.34 (m, H-9), 1.05−0.95 (m, H-11), 0.89 (t, J = 7.4 Hz, H-10, 5S,8S), 0.80 (t, J = 7.4 Hz, H-10, 5R,8S). Third fraction (1): 1H NMR (400 MHz CDCl3) δ 3.78 (s, H-5, 5S,8S), 2.51 (s, H-7, 5S,8S), 2.01−1.88 (m, H-8), 1.46−1.16 (m, H-9), 1.00 (d, J = 7.0 Hz, H-11, 5S,8S), 0.90 (t, J = 7.4 Hz, H-10, 5S,8S). Calibration. The stock solutions of 1/2 and 13C2-1/13C2-2 are both mixtures of both isomers with equal concentrations of 1/2, which was confirmed by NMR16 and by HPLC-MS/MS. Therefore, the stock solution containing 10.97 μg/mL of both isomers contains 5.49 μg/ mL of each isomer, and the isotopically labeled stock solution contains 28.15 μg/mL of each labeled isomer. Solutions of 1/2 and the labeled standards were evaporated in a nitrogen stream to obtain nine calibration points ranging from 5.5 to 517 ng/mL of both 1 and 2, respectively. Each calibration solution contained 106 ng/mL of both labeled standards, respectively. The evaporated standards were resuspended in the injection solvent (MeCN/H2O/FA, 10:89:1, v/ v/v). Samples. Tomato products (tomato puree and juice, ketchup, minced tomatoes, tomato-based pasta sauce) were bought in the region of Münster, Germany. The samples were transferred to 50 mL polypropylene tubes and stored at −20 °C. Fungal Cultures. Four different strains of A. alternata were cultured in a liquid YES medium (yeast extract sucrose medium; 20 g/ L yeast extract and 150 g/L sucrose). Three strains (MRI 1275, 1277, and 1293) were obtained from the Max Rubner-Institute (Karlsruhe, Germany), and one strain (DSMZ 12633) was purchased from the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Two hundred milliliters of medium was autoclaved in 500 mL Erlenmeyer flasks and inoculated with approximately 0.25 cm2 of agar plates covered with the respective strain. The flasks were sealed with metal caps and incubated at 28 °C for 14 days in the dark. After 7 and 14 days, respectively, 1 mL of each liquid culture was filtered through a rectified cellulose, 0.45 mm, syringe filter (Phenomenex, Aschaffenburg, Germany) and analyzed immediately. The strains were also cultured in tomato puree free of 1 and 2 above the LOD. Two hundred milliliters of the puree was autoclaved in Erlenmeyer flasks as described above. One hundred microliters of the liquid cultures in YES medium after 7 days of incubation was transferred to the autoclaved tomato purees. The puree was incubated at 28 °C in the dark for 10 days. The cultures were homogenized using a blender, and the pH values of both media (YES and tomato puree) were measured before inoculation and after 14 days (YES medium) or
MATERIALS AND METHODS
Chemicals and Reagents. All solvents used were of gradient grade and, if not stated otherwise, purchased from VWR (Darmstadt, Germany). HPLC-MS grade methanol was purchased from Fisher Scientific (Loughborough, UK). Water was purified with a Milli-Q Gradient A10 system from Millipore (Schwalbach, Germany). Formic acid was purchased from Grüssing (Filsum, Germany), ammonium acetate (NH4OAc) from Sigma-Aldrich (Steinheim, Germany), magnesium sulfate (MgSO4) from Carl Roth (Karlsruhe, Germany), and sodium chloride (NaCl) from Merck (Darmstadt, Germany). Polypropylene tubes (15 and 50 mL) were provided by Greiner Bio One (Frickenhausen, Germany). 1 and 2 as a racemic mixture as well as 13C2-1 and 13C2-2 as a racemic mixture were synthesized in our work group.16 (See Hickert et al.23 for NMR data for unlabeled 1/2.) 1 (1 mg) as a reference substance was purchased from Cfm Oskar Tropitzsch GmbH (Marktredwitz, Germany). A tomato puree free of 1 and 2 above the LOD of this method was prepared by blending tomatoes purchased in a local grocery store. The puree was passed through a metal sieve (0.5 mm mesh size) and stored at −20 °C. UV Spectroscopy. There are several molar absorptivity values for 1 published,2,21 but not for the solvent acetonitrile. We determined a molar absorptivity value for 1 and 2 in acetonitrile by weighing approximately 10 mg of the racemic mixture of 1 and 2 in a glass flask. The flask was covered with perforated Parafilm and subjected to lyophilization to remove any possibly remaining water. The exact weight was recorded, and 10 mL of MeCN was added to the flask. The flask was subsequently placed in an ultrasonic bath for 30 min. The resulting stock solution was diluted with MeCN to 8.90 μg/mL in quintuplicate, and UV−vis spectra in a range of 190−400 nm were recorded. The absorptivity value was calculated at the absorption maximum of 276 nm using the equation ε = (absorption × 1000)/ (concentration in mmol/L). A molar absorptivity value for the racemic mixture of 1 and 2 of (1.427 ± 0.016) × 104 L/mol/cm at 276 nm in acetonitrile was calculated. Values of 4.13 log e (which equals 1.349 × 104 L/mol/cm) in ethanol at 277 nm2 and of 1.298 × 104 L/mol/cm in methanol at 277 nm21 are described in the literature. These molar absorptivity values are in good accordance with the value determined in this study. Preparation of Standard Solutions. The racemic mixtures of 1 and 2 as well as 13C2-1 and 13C2-2 were both dissolved in MeCN and the exact concentrations determined by UV spectroscopy on a DU 800 spectrometer (Beckman Coulter GmbH, Krefeld, Germany) using the molar absorptivity value of (1.427 ± 0.016) × 104 L/mol/cm at 276 nm in MeCN determined in this study. Stock solutions of 10.97 μg/mL 1/2 and 56.30 μg/mL 13C2-1/13C22 were obtained. The purchased reference substance was dissolved in MeCN to obtain a solution with 250 μg/mL 1. 10880
DOI: 10.1021/acs.jafc.5b04812 J. Agric. Food Chem. 2015, 63, 10879−10884
Article
Journal of Agricultural and Food Chemistry 10 days (tomato puree) (pH-meter 766 Calimatic, Knick Elektronische Messgeräte GmbH & Co. KG, Berlin, Germany) and found to be pH 4.2 for tomato puree and pH 6.5 for YES medium before inoculation. The pH values in YES medium did not change after incubation, but in tomato puree, the pH values varied (MRI 1275, pH 3.2; MRI 1277, pH 4.6; MRI 1293, pH 4.4; DSMZ 12633, pH 6.7; Table 1). Portions of the contaminated tomato purees were worked up analogously to the food samples and analyzed by HPLC-MS/MS.
tomato paste was prepared to investigate whether the low pH value of the tomato puree (pH 3.2−6.7; depending on strain) in combination with elevated temperatures may lead to an epimerization from 1 to 2 as observed in aqueous solutions (pH 3.5).12 The naturally contaminated tomato purees were heated to 65 °C24 and stirred on a heating block for 90 min. The purees were cooled immediately afterward using ice and analyzed as described for the food samples. Sample Preparation. A modified, QuEChERS-based sample preparation16 described by Lohrey et al. was applied. Tomato products (2.5 ± 0.05 g) were weighed in a 50 mL polypropylene tube, and the exact sample weights were recorded. Fifty microliters of a solution of the labeled standards in MeCN (11.3 μg/mL of each isomer) was added followed by 10 mL of water. The samples were shaken on a vortex shaker for 30 s, and 10 mL of MeCN (1% FA, v/v) was added. The extraction was carried out on a laboratory shaker for 25 min. Five grams (±0.1 g) of a salt mixture (MgSO4/NaCl, 4:1, m+m) was poured in the polypropylene tubes, and the tubes were again shaken for 30 s and subsequently centrifuged at 3500g for 5 min. During centrifugation, phase separation between aqueous and organic phases took place. Six milliliters of the upper organic layer was transferred to a 15 mL polypropylene tube already containing 1.2 (±0.1) g of MgSO4. The tubes were again shaken for 30 s, and after centrifugation (3500g, 5 min), 1 mL of the supernatant was transferred to a glass vial, evaporated to dryness in a nitrogen stream, resuspended in the injection solution (300 μL, MeCN/H2O/FA, 10:89:1, v/v/v), and centrifuged at 3500g for 10 min. Two hundred microliters of the clear supernatant was transferred to a glass insert and the insert placed in
Table 1. Concentrations of 1 and 2 in Tomato Purees after Inoculation with Different Alternaria alternata Strains and after Heat Treatment
strain DSMZ 12633 MRI 1275 MRI 1277 MRI 1293
content of 1 after 10 days (μg/kg)
content of 1 after heat 2 after treatment 10 days (μg/kg) (%)
2 after heat treatment (%) 3.4
pH of tomato puree after 10 days of incubation
440
1100
3.3
150
LOQ 4.2 12 1.4 17 160 16 120 5.2 20
± ± ± ± ± ± ± ± ±
0.3 0.2 0.4 3.4 4.8 0.6 5.4 0.6 2.9
8.0
n. d. 7.0 21 17 33 38 28 36 14 23
of the analyzed purees after heat treatment (Table 2). 2 was found in a higher percentages in processed tomato products than in tomato puree inoculated with A. alternata after 10 days. This can be explained by the longer shelf life of commercial tomato products. Occurrence of 1 and 2 in Tomato Products. Twenty tomato products from the German market were analyzed by stable-isotope dilution HPLC-MS/MS for 1 and 2. All samples were contaminated with both isomers. The results are summarized in Table 2. 1 was found in a range from 5.3 ± 0.1 to 550 ± 15 μg/kg (average = 120 μg/kg) and 2 in a range from 1.4 ± 0.4 to 270 ± 0.8 μg/kg (average = 57 μg/kg). 2 represents 7.0−44% of the sum of both isomers (average = 29%). 1 could be quantitated in all samples and 2 in 19 of 20 samples. Only in one sample did the signal for 2 range between the LOD (0.29 μg/kg) and the LOQ (0.97 μg/kg). This is the first report of the occurrence of 2 in food samples. It has been demonstrated that no epimerization of 1 to 2 or vice versa takes place under the sample preparation conditions used. Differences in the toxicity of 1 and 2 in cell culture models have been shown in this study, and 2 represents a major portion of the sum of both isomers (29% on average). Methods quantitating both isomers as a sum total may overestimate the risk posed by 1 in food samples. Further research on the toxicity of 2 in vitro and in vivo as well as on the epimerization during food processing is needed to get a clear picture of the differences in toxicity of both substances and epimerization conditions. In conclusion, we could for the first time detect 2, the epimer of 1, together with 1 in all 20 analyzed commercial tomato products. 1 and 2 were tested for their cytotoxic effects on HT-
Figure 3. Cell viability of HT-29 cells after incubation with 1 and 2 for 48 h. The results represent the sum of three individual experiments each with three triplicates (n = 9, 2) and four individual experiments each with three triplicates (n = 12, 1). (∗) Statistically significant (p ≤ 0.05); (∗∗) statistically highly significant (p ≤ 0.01).
250 to 800 μM, 1 exhibits statistically highly significant (∗∗) reduction of cell viability. For 2 no decrease in cell viability was detectable in the analyzed concentration range (10−800 μM, Figure 3). Therefore, in contrast to 1, 2 did not show any cytotoxic properties in HT-29 cells. Analysis of Fungal Cultures for 1 and 2. Four different strains of A. alternata (MRI 1275, MRI 1277, MRI 1293, and DSMZ 12633) were cultured in liquid YES medium and in tomato puree. Although no 2 could be detected in the YES medium, small percentages of 2 could be detected in tomato puree (Table 2). The pH value of the YES medium was close to neutral before and after cultivation (6.5), whereas the pH values of the tomato puree changed during cultivation. As predicted by Siegel et al.,12 the percentage of 2 in the tomato puree was influenced by the pH value of the culture medium after cultivation. The most acidic culture (pH 3.2) contained the highest percentage of 2 (4.2%). During heat treatment, 1 was completely degraded in the most acidic sample, while the concentrations increased, due to the evaporation of water, in the other samples. The percentage of 2 did not increase in any 10883
DOI: 10.1021/acs.jafc.5b04812 J. Agric. Food Chem. 2015, 63, 10879−10884
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Journal of Agricultural and Food Chemistry
(12) Siegel, D.; Merkel, S.; Bremser, W.; Koch, M.; Nehls, I. Degradation kinetics of the Alternaria mycotoxin tenuazonic acid in aqueous solutions. Anal. Bioanal. Chem. 2010, 397, 453−462. (13) Lebrun, M. H.; Nicolas, L.; Boutar, M.; Gaudemer, F.; Ranomenjanahary, S.; Gaudemer, A. Relationships between the structure and the phytotoxicity of the fungal toxin tenuazonic acid. Phytochemistry 1988, 27, 77−84. (14) Chen, S.; Xu, X.; Dai, X.; Yang, C.; Qiang, S. Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii, Biochim. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 306−318. (15) Gitterman, C. O. Antitumor, cytotoxic and antibacterial activities of tenuazonic acid and congeneric tetramic acids. J. Med. Chem. 1965, 8, 483−486. (16) Lohrey, L.; Marschik, S.; Cramer, B.; Humpf, H.-U. Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products. J. Agric. Food Chem. 2013, 61, 114−120. (17) Asam, S.; Liu, Y.; Konitzer, K.; Rychlik, M. Development of a stable isotope dilution assay for tenuazonic acid. J. Agric. Food Chem. 2011, 59, 2980−2987. (18) Gross, M.; Curtui, V.; Ackermann, Y.; Latif, H.; Usleber, E. Enzyme immunoassay for tenuazonic acid in apple and tomato products. J. Agric. Food Chem. 2011, 59, 12317−12322. (19) Siegel, D.; Rasenko, T.; Koch, M.; Nehls, I. Determination of the Alternaria mycotoxin tenuazonic acid in cereals by highperformance liquid chromatography-electrospray ionization ion-trap multistage mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. J. Chromatogr. A 2009, 1216, 4582−4588. (20) Lebrun, M. H.; Gaudemer, F.; Boutar, M.; Nicolas, L.; Gaudemer, A. Ion-pair, anion-exchange and ligand-exchange highperformance liquid chromatography of tenuazonic acid and 3-acetyl 5substituted pyrrolidine-2,4-diones. J. Chromatogr. A 1991, 464, 307− 322. (21) Scott, P. M.; Kanhere, S. R. Liquid chromatographic determination of tenuazonic acids in tomato paste. J.−Assoc. Off. Anal. Chem. 1980, 63, 612−621. (22) Tö lgyesi, Á .; Stroka, J.; Tamosiunas, V.; Zwickel, T. Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimized method using liquid chromatography−tandem mass spectrometry. Food Addit. Contam., Part A 2015, 32, 1512−1522. (23) Hickert, S.; Bergmann, M.; Ersen, S.; Cramer, B.; Humpf, H.-U. Survey of Alternaria toxin contamination in food from the German market, using a rapid HPLC-MS/MS approach. Mycotoxin Res. 2015, 10.1007/s12550-015-0233-7 (24) Zanoni, B.; Pagliarini, E.; Giovanelli, G.; Lavelli, V. Modelling the effects of thermal sterilization on the quality of tomato puree. J. Food Eng. 2003, 56, 203−206. (25) Ishiyama, M.; Miyazono, Y.; Sasamoto, K.; Ohkura, Y.; Ueno, K. A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 1997, 44, 1299− 1305. (26) Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. A water soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 1999, 36, 47−50. (27) Mulac, D.; Humpf, H.-U. Cytotoxicity and accumulation of ergot alkaloids in human primary cells. Toxicology 2011, 282, 112− 121. (28) Annesley, T. M. Ion suppression in mass spectrometry. Clin. Chem. 2003, 49, 1041−1044.
29 cells; 2 had no toxic effects in the concentration range investigated (10−800 μM), whereas 1 highly significantly decreased the cell viability in a concentration-dependent manner starting at 250 μM (p ≤ 0.01). Small amounts of 2 were produced by A. alternata in tomato puree.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04812. SRM transitions for 1, 2, 13C2-1, and 13C2-2 including parent ion masses, fragment ion masses, ion ratios, retention times, declustering potentials, collision energies and collision cell exit potentials, HPLC-MS/MS chromatograms of 12.5 ng/mL 1 in the presence of 237.5 ng/mL 2 and vice versa, and a HPLC-MS/MS chromatogram of isomerically pure 1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(H.-U.H.) Phone: +49 251 33391. Fax: +49 251 83 33396. Email:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Angela Klusmeier-König and Steffen Lürwer for support during sample preparation. REFERENCES
(1) Rosett, T.; Sankhala, R. H.; Stickings, C. E.; Taylor, M. E. U.; Thomas, R. Metabolites of Alternaria tenuis auct.: culture filtrate products. Biochem. J. 1957, 67, 390−400. (2) Stickings, C. E. Metabolites of Alternaria tenuis auct.: the structure of tenuazonic acid. Biochem. J. 1959, 72, 332−340. (3) Ostry, V. Alternaria mycotoxins: an overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J. 2008, 1, 175−188. (4) European Food Safety Authority (EFSA) Scientific Opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food, EFSA J. 2011, 9 (10), 1−97. (5) Pero, R. W.; Posner, H.; Blois, M.; Harvan, D.; Spalding, J. W. Toxicity of metabolites produced by the “Alternaria”. Environ. Health Perspect. 1973, 4, 87−94. (6) Griffin, G. F.; Chu, S. S. Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay. Appl. Environ. Microbiol. 1983, 46, 1420− 1422. (7) Youngner, J. S. Influence of inhibitors of protein synthesis on interferon formation in mice. Virology 1970, 40, 335−343. (8) Smith, E. R.; Fredrickson, T. N.; Hadidian, Z. Toxic effects of the sodium and the N,N′-dibenzylethylenediamine salts of tenuazonic acid (NSC-525816 and NSC-82260). Cancer Chemother. Rep. 1968, 52, 579−585. (9) Joshi, A.; Min, Z.; Brumley, W. C.; Dreifuss, P. A.; Yang, G. C.; Sphon, J. A. Mass spectrometry of the copper salt of tenuazonic acid. Biomed. Mass Spectrom. 1984, 11, 101−105. (10) Shephard, G. S.; Thiel, P. G.; Sydenham, E. W.; Vleggaar, R.; Marasas, W. F. O. Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids. J. Chromatogr., Biomed. Appl. 1991, 566, 195−205. (11) Gallardo, G. L.; Peña, N. I.; Chacana, P.; Terzolo, H. R.; Cabrera, G. M. L-Tenuazonic acid, a new inhibitor of Paenibacillus larvae. World J. Microbiol. Biotechnol. 2004, 20, 609−612. 10884
DOI: 10.1021/acs.jafc.5b04812 J. Agric. Food Chem. 2015, 63, 10879−10884
