Article pubs.acs.org/JAFC
Natural Occurrence of Four Alternaria Mycotoxins in Tomato- and Citrus-Based Foods in China Kai Zhao,†,‡ Bing Shao,§ Dajin Yang,‡ and Fengqin Li*,‡ †
Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, 7 Panjiayuan Nanli, Chaoyang District, Beijing 100021, People’s Republic of China ‡ Key Laboratory of Food Safety Risk Assessment, Ministry Health, China National Center for Food Safety Risk Assessment, 7 Panjiayuan Nanli, Chaoyang District, Beijing 100021, People’s Republic of China § Beijing Center for Disease Control and Prevention, 16 Hepingli Middle Street, Dongcheng District, Beijing 100013, People’s Republic of China S Supporting Information *
ABSTRACT: A total of 70 tomato-based and 86 citrus-based products collected in China were analyzed for alternariol, alternariol monomethyl ether, tentoxin, and tenuazonic acid by ultraperformance liquid chromatography−electrospray ionization−tandem mass spectrometry. No toxins were found in any fresh tomato or citrus fruit samples. Tenuazonic acid was the predominant toxin detected in all tomato ketchup (10.2−1787 μg/kg) and tomato juice samples (7.4−278 μg/kg). Alternariol was quantitated at higher level than alternariol monomethyl ether with the ratio of alternariol/alternariol monomethyl ether ranging from 0.37 to 104 in 14 alternariol-positive tomato ketchup samples. Tentoxin was detected at much lower levels in all samples analyzed. Some citrus juice samples were positive for tenuazonic acid and alternariol monomethyl ether. It is necessary to conduct a systemic surveillance of Alternaria toxins in raw and processed foods to provide the scientific basis for risk assessment of dietary exposure to these toxins in Chinese populations. KEYWORDS: Alternaria toxins, tomato-based products, citrus-based products, China, UPLC-MS/MS
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INTRODUCTION Alternaria toxins are produced by Alternaria species that are saprophytic and pathogenic on many plants. These species of fungi produce more than 70 secondary metabolites, but a small proportion of these toxins have been chemically characterized and documented to act as mycotoxins to humans and animals.1,2 The most important Alternaria toxins with toxicological significance reported to occur in cereals, oilseeds,3 vegetables (especially tomato),4,5 and fruits (apples,6 citrus fruits7 etc.) are alternariol, 1; alternariol monomethyl ether, 2; tentoxin, 3; and tenuazonic acid, 4 (Figure 1). Limited in vivo study showed that alternariol monomethyl ether poses fetotoxicity and teratogenicity to hamsters.8 Moreover, alternariol and alternariol monomethyl ether are mutagenic and genotoxic in various in vitro systems, and there is also some evidence for carcinogenic properties in unconventional assays.9,10 In addition, it has been suggested that the contamination of Alternaria toxins in small grains, especially in wheat grains, is much heavier in the high-incidence areas for human esophageal cancer than in the low-incidence areas.11 The production of these toxins is affected by interactions among the Alternaria strain, the growing substrate, and the environmental conditions.2 Fresh plant products affected by Alternaria regularly present visible rotten areas, and the Alternaria toxins can be transferred from the rotten part to the surrounding tissues.6 Hence, Alternaria toxins are also present in processed plant products due to the limitations of the current industrial procedures to eliminate the rotting tissues completely.12 The occurrence of Alternaria mycotoxins in © XXXX American Chemical Society
processed foodstuffs is becoming an increasing concern for public health. The analytical methods for the analysis of these toxins have focused on liquid chromatography coupled to single mass spectrometry (MS) or tandem MS.7,10,13−17 Stable isotope dilution assays have been employed in the determination of Alternaria toxins, in which labeled internal standards were used to compensate for analyte losses during sample preparation and ion suppression in the ESI interface in current methodology.16,18,19 Tomatoes and citrus fruits can easily be infected by fungi of Alternaria, and their products such as tomato ketchups and citrus juice are frequently contaminated with Alternaria mycotoxins.7 Although only a limited number of papers describe the contamination of Alternaria toxins in local agricultural crops in China, the natural occurrence of these mycotoxins in tomato and tomato products, citrus, and citrus products have not been reported so far. On the other hand, risk assessments relating to food safety in China are frequently hampered by the lack of quantitative data. According to the European Food Safety Authority (EFSA),10 the contribution to dietary exposure to Alternaria toxins is mainly made by grain and grain-based products, vegetables and vegetable products, and fruits and fruit products, depending on the toxin and the food consumption pattern in different countries. To the present Received: July 18, 2014 Revised: December 12, 2014 Accepted: December 18, 2014
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DOI: 10.1021/jf5052738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of tenuazonic acid, 1; alternariol, 2; tentoxin, 3; and alternariol monomethyl ether, 4. methanol−water (20:80, v/v). Alternaria toxins were eluted with 5 mL of methanol and 5 mL of acetonitrile sequentially. The organic phase elutes were combined, evaporated to dryness, and dissolved in 1 mL of methanol/water (10:90, v/v) containing 0.5 mmol/L ammonium bicarbonate and centrifuged for 5 min at 10000 rpm. An aliquot of 10 μL of the extract was injected for Alternaria toxins analysis by UPLCMS/MS. UPLC Conditions. Detection and quantitation of the four target mycotoxins were performed on a Micromass Quattro Premier XE ultra performance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS) system (Waters, Milford, MA, USA). The column used was a 100 mm × 2.1 mm i.d., 1.7 μm, BEH C18 (Waters) thermostated at 40 °C. The mobile phase included 0.5 mmol/L ammonium bicarbonate solution (solvent A) and methanol (solvent B). A binary gradient at a flow rate of 0.3 mL/min was programmed starting at 90% A, reached 100% B in 5 min, and was maintained there for 2 min. Afterward, B was linearly decreased to 10% B within 0.1 min and maintained at this composition of mobile phase for 2 min. The retention times of the four toxins were 1.69 min for tenuazonic acid, 3.79 min for alternariol, 4.38 min for tentoxin, and 4.94 min for alternariol monomethyl ether. Preparation of Standard Solutions. Stock Alternaria toxin standard solutions at the concentration of 100 mg/L were prepared by dissolving appropriate amounts of alternariol, alternariol monomethyl ether, tentoxin, and tenuazonic acid standard powder in methanol, respectively, and stored in amber vessels at −18 °C for up to 1 year. Working standard solutions were made by diluting the stock standard solutions with Alternaria toxin-free sample extract prepared following the toxin extraction procedure. MS/MS Conditions. MS/MS was performed on a Waters Aquity Quattro Premier XE triple-quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. The source block temperature was 100 °C, and desolvation temperature was 350 °C. Nitrogen (purity = 99.9%) was used as both the cone gas and desolvation gas at flow rates of 1 and 552 L/h, respectively, whereas high-purity argon was used as the collision gas at a flow rate of 0.2 mL/min. The mass spectrometer was operated in negative electrospray ionization (ESI−) mode for quantitation of the four toxins. Data acquisition and evaluation were performed by Masslynx software (Micromass, Manchester, UK). Method Validation. To evaluate the performance of UPLC-MS/ MS conditions for analysis of the four mycotoxins, the matrix-matched calibration curve was made to compensate for matrix effect. Standard solutions of the four mycotoxins at concentrations ranging from 5 to 100 μg/L for tenuazonic acid, from 20 to 400 μg/L for alternariol, from 2 to 40 μg/L for tentoxin, and from 0.4 to 8 μg/L for alternariol monomethyl ether were added to the final extract solution of mycotoxin-free fresh tomato, tomato ketchup, fresh citrus, and citrus juice and processed for analysis by LC-MS/MS. To determine the method recoveries and the relative standard deviation (RSD), analyses of the spiked four matrix samples at spiking levels between 5 and 50 μg/kg (six repetitions for each) for the four mycotoxins were carried out. The limit of detection (LOD) and the limit of quantification (LOQ) for these four mycotoxins were determined with signal-tonoise ratios of 3:1 and 10:1, respectively. Reproducibility was evaluated over five consecutive days at three nominal tenuazonic acid concentrations (50, 500, and 5000 μg/kg) by spiking the
time, however, no study has been carried out for Alternaria toxin contamination in tomato and citrus products at the national level in China. Therefore, no data on risk assessment of dietary exposure to Alternaria toxins in the Chinese population is available so far. To provide the scientific data for assessing the impact of Alternaria toxins on Chinese public health, the purpose of this study was to evaluate the natural occurrence of associated Alternaria mycotoxins in Chinese fresh tomato, tomato ketchup, and fresh citrus, and citrus juice. The results obtained will be expected to contribute to either the development of strategies to reduce risk from these contaminants or the ongoing research on the relationship between the consumption of tomato and citrus products contaminated with Alternaria toxins and human disease.
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MATERIALS AND METHODS
Reagents and Chemicals. Methanol and acetonitrile used for sample extraction and toxin separation were of HPLC grade (Dikma Pure, Richmond Hill, GA, USA). Both ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and formic acid (≥95% purity) (Fluka, Steinheim, Germany) were of analytical grade. Pure water was obtained from a Millipore Milli-Q System (Millipore, Bedford, MA, USA). Standards for alternariol (purity > 98%), alternariol monomethyl ether (purity > 98%), tentoxin (purity > 99%), and tenuazonic acid (purity > 99%) were purchased from Fermenteck Ltd. (Jerusalem, Israel). All experimental practice followed Environmental Health Safety Guidelines for the use of chemicals authorized by China National Center for Food Safety Risk Assessment. In addition, sample collection and solvent extracts should be handled with care. Samples. A total of 30 fresh tomato and 40 tomato-based products including tomato ketchup (n = 31) and tomato juice (n = 9) samples as well as 50 fresh citrus and 36 citrus juice samples were randomly collected from supermarkets and agricultural trade markets in Beijing. The fresh tomatoes covered the main cultivars in China such as common tomato and cherry tomato that came from the main vegetable-producing regions including Beijing, Shandong, Hebei, Jiangsu, Tianjin, Fujian, Zhejiang, and Guangdong provinces. Tomato ketchup and tomato juice samples covered the main brands available in markets in China; the nine tomato ketchup samples were imported from Bulgaria (two samples), Italy (two samples), Japan, Turkey, Australia, the United States, and Spain, respectively. All samples were stored at 4 °C until analysis. Toxin Extraction. The extraction of Alternaria toxins from samples was modified on the basis of the methods described by Kocher20 and Rheinhold et al.21 with a minor modification. In brief, both fresh tomatoes and citrus fruits were fully ground, whereas tomato- and citrus-based products were blended or shaken thoroughly. A test portion of 5 g of sample was homogenized with 20 mL of acetonitrile/ water/methanol (45:45:10, v/v/v) and sonicated in an ultrasonic cleaner for 30 min, followed by centrifuging for 10 min at 7000 rpm. A portion of 5 mL of supernatant was diluted 5 times with distilled water, adjusted to pH 3−4 with formic acid, and passed through a HLB solid phase extraction (SPE) cartridge (Waters, Rupperswil, Switzerland) preconditioned with 5 mL of methanol followed by 5 mL of water. The cartridge was washed with 5 mL of water followed by 5 mL of B
DOI: 10.1021/jf5052738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry uncontaminated matrix. For each concentration and day, the standard addition scheme described above was conducted. For determination of intralaboratory reproducibility, two tomato ketchup samples naturally contaminated with tenuazonic acid, alternariol, tentoxin, and alternariol monomethyl ether at levels of 370, 37, 3.3, and 10 μg/kg and two citrus juice samples naturally contaminated with tenuazonic acid, alternariol, tentoxin, and alternariol monomethyl ether at concentrations of 370, 37, 3.3, and 10 μg/kg, respectively, were analyzed three times within 2 weeks, and duplicate analyses were conducted for each sample each time.
in tomato ketchup and 4.2% in citrus juice, 14.6% for alternariol in tomato ketchup and 14.6% in citrus juice, 12.3% for tentoxin in tomato ketchup and 12.3% in citrus juice, and 9.8% for alternariol monomethyl ether in tomato ketchup and 9.8% in citrus juice. On the basis of these results, we conclude that the extraction efficiencies achieved by the method used in the present study were high and consistent. Under LC-MS/MS conditions, very good resolution for the four mycotoxins in tomato samples was observed and their spectra were similar to those of the corresponding standards. Natural Occurrence of the Four Alternaria Toxins in Fresh Tomato and Tomato-Based Products. Contamination levels of the four Alternaria toxins in Chinese tomato and tomato-based product samples are given in Table 1. No
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RESULTS AND DISCUSSION Method Optimization. For the four toxins cleanup from a HLB SPE cartridge, methanol/water in different ratios (20:80, 30:70, and 40:60, v/v) and acetonitrile/water/methanol (45:45:10, v/v/v), respectively, were studied. Better cleanup efficiency of the analytes was obtained when methanol/water (20:80, v/v) was selected. Additionally, the choice of mobile phase should be based on the consideration of ionization efficiency before the analytes enter the MS/MS system to obtain good resolution and high sensitivity. In terms of additives in the mobile phase, aqueous ammonia, ammonium formate, and ammonium bicarbonate were studied. Responses and peak shape were greatly improved when 0.5 mmol/L of aqueous solution of bicarbonate was used as solvent A. Results indicated that when acetonitrile was chosen as the mobile phase, the ionization of all the selected analytes was significantly suppressed, and the abundance and sensitivity were thus reduced. Conversely, very positive results were observed when methanol was used as the mobile phase. Therefore, methanol was selected as the strong elution mobile phase in the present study. Method Validation. Matrix-matched standards were used for calibration to compensate for the matrix effect. The concentrations used for making calibration curves ranged from 5 to 100 μg/L for 1, from 20 to 400 μg/L for 2, from 2 to 40 μg/L for 3, and from 0.4 to 8 μg/L for 4 with linear coefficients better than 0.999. The method showed an excellent linearity over the ranges of 16−320 μg/kg for 1, 0.3−6 μg/kg for 2, 1.6−32 μg/kg for 3, and 4−80 μg/kg for 4 expressed by the coefficients (r2) of >0.99 for all curves. The validation results demonstrated high sensitivity, repeatability, and linearity of the method. The LODs of tenuazonic acid, alternariol, tentoxin, and alternariol monomethyl ether were 1, 5, 0.5, and 0.1 μg/kg in tomato-based products and 1, 6, 0.7, and 0.1 μg/kg in citrusbased samples, respectively, which were the same as or lower than those reported by Noser et al.5 but higher than those obtained with the stable isotope method.14,16,19 To extract the four mycotoxins rapidly from the tested matrix and to eliminate coextraction of potentially interfering matrix components as much as possible, absolute recoveries of the four toxins from four matrices were assessed. The recovery of tenuazonic acid from tomato ketchup was performed with a naturally tenuazonic acid-contaminated sample because no tenuazonic acid-free tomato ketchup samples were found in this study. For each analyte/matrix combination, the recoveries at three spiking levels ranged from 73.4 to 111.2% for all four toxins in tomato- and citrus-based product samples; values fall within the range of 70−120% as recommended by the European Commission.22 On the other hand, RSDs for all four toxins were alternariol > alternariol monomethyl ether > tentoxin.7 In particular, alternariol was quantitated at higher levels than alternariol monomethyl ether with the ratio of alternariol/alternariol monomethyl ether up to 104 (average = 17) in 14 alternariol-positive samples. These results tended to be different from those in wheat reported previously in China.23 Additionally, a positive correlation between alternariol and alternariol monomethyl ether (r2 = 0.850, P < 0.001) and also the total dibenzopyrone derivatives (alternariol + alternariol monomethyl ether) and tenuazonic acid (r2 = 0.796, P < 0.001) in wheat samples has been demonstrated by Li et al.,23 indicating the coproduction of these toxins on wheat grains by Alternaria species in fields. However, no significant correlations were found in concentrations between alternariol and alternariol monomethyl ether (r2 = 0.070) and total dibenzopyrone derivatives and tenuazonic acid (r2 = 0.035) in tomato ketchup samples. Invasion of tomato and wheat by different species of Alternaria may mainly contribute to the difference in toxin levels in the same area. In contrast, alternariol was found at levels of 1 and 13 μg/kg with high frequency in apple (67% of 44 samples) and tomato (93% of 44 samples) products, respectively, reported by Ackermann et al.24 A survey conducted in Brazil revealed that neither alternariol monomethyl ether nor alternariol was detected in 80 samples, but tenuazonic acid was found in 7 tomato pulp (39−111 μg/kg) and 4 tomato puree (29−76 ng/ g) samples.25 Low mean levels of alternariol (up to 25 μg/kg) and alternariol monomethyl ether (up to 0.7 μg/kg) were measured in vegetables, mainly tomato paste.16 Tenuazonic acid at levels between 15 and 195 μg/kg, between 363 and 909 μg/kg, and between 8 and 242 μg/kg were found in commercial tomato ketchup, tomato paste, and pureed tomato samples analyzed by stable isotope dilution assays, respectively.16 All results mentioned above are much lower than ours. However, about 60% of Argentinian tomato pulp samples were contaminated with tenuazonic acid, alternariol monomethyl ether, and alternariol at levels up to 4021 μg/kg (29% of 80 samples), 1734 μg/kg (26% of 80 samples), and 8756 μg/kg (6% of 80 samples),4 respectively, with the maximum levels of tenuazonic acid, alternariol, and alternariol monomethyl ether higher than those obtained in the present study. Alternaria mycotoxins in tomato and citrus products are most likely to come from the contaminated raw material. Tomato ketchup was made from tomatoes through a series of steps including washing, heating, pulping, condensing, etc. It is wellknown that Alternaria rapidly colonizes tomatoes and produces mycotoxins under favorable conditions. It was speculated that Alternaria mycotoxins in tomato ketchup probably came from decayed tomatoes or from the steps contaminated with Alternaria species during the production line.4 Alternaria mycotoxins detected at higher concentrations in tomato ketchup than in fresh tomatoes were partly due to the former being concentrated after production.
EFSA has given a scientific opinion on the risks for animal and public health related to dietary exposure to seven Alternaria mycotoxins.10 The estimated chronic dietary exposures for the four toxins were much lower than the corresponding threshold of toxicological concern (TTC), indicating no human health concern according to the in vitro model for intestinal absorption and consumption data in China (data not shown).26,27 However, alternariol and alternariol monomethyl ether were genotoxic and bound to the DNA of esophageal epithelium of the human fetus, activated oncogenes in the human fetal epithelium, and induced epithelial proliferation of human fetal esophagus in vitro.28 Although Alternaria spp. are the most frequent fungal species invading tomatoes,2,12 there are no specific regulations for any of the Alternaria toxins in foods in China so far. Therefore, systemic long-term surveillance of Alternaria toxins in raw and processed foods all over China, especially in the area with high incidence of esophageal cancer, as well as a thorough assessment of dietary exposure to these toxins in Chinese populations should be intensified to evaluate consumers’ health risk.
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ASSOCIATED CONTENT
S Supporting Information *
Tables A−C (MRM parameters, LODs and LOQs, and recoveries and RSDs of the four toxins in samples). This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(F.L.) Phone/fax: 86-10-67776356. E-mail: lifengqin@cfsa. net.cn. Funding
This study was financially supported by the National High-tech R&D Program (863 Program) and the Ministry of Science and Technology of the People’s Republic of China (Grant 2012AA101603). Notes
The authors declare no competing financial interest.
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REFERENCES
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