Article Cite This: J. Agric. Food Chem. 2018, 66, 11846−11853
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Determination of Alternaria Mycotoxins in Fresh Sweet Cherries and Cherry-Based Products: Method Validation and Occurrence Xiaoting Qiao,† Jie Yin,‡ Yunjia Yang,‡ Jing Zhang,‡ Bing Shao,*,†,‡ Hui Li,‡ and Hongbing Chen† †
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P.R. China Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control, Beijing 100013, P.R. China
J. Agric. Food Chem. 2018.66:11846-11853. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 11/08/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Sweet cherry is susceptible to disease caused by the Alternaria species and produces various Alternaria mycotoxins. Analytical methodologies based on solid-phase extraction (SPE) and LC-MS/MS to simultaneously determine five main Alternaria mycotoxins (tenuazonic acid, 1; alternariol, 2; alternariol methyl ether, 3; altenuene, 4; and tentoxin, 5) in fresh sweet cherries and cherry products were developed and validated. The limits of quantitation (LOQ) of the analytes ranged from 0.002−0.066 μg/kg. The method was successfully applied to 83 fresh cherry and cherry-related product samples. 1 and 5 were the predominant toxins with detection frequencies >50%, followed by 3 (42%), 2 (35%), and 4 (31%). Daily intakes of Alternaria mycotoxins via fresh sweet cherries were assessed preliminarily using the measured concentrations, and consumption data were obtained from a web-based dietary questionnaire (n = 476). The maximum exposure of 1 and 3 were 4.6 and 16.7 times the threshold of the toxicological concern (TTC) value, respectively. KEYWORDS: Alternaria mycotoxins, UPLC-MS/MS, sweet cherry, cherry products
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INTRODUCTION Alternaria species are ubiquitous microfungi in soil, wallpapers, textiles, and even air.1 Their widespread occurrence and ability to grow at low temperatures make them one of the most important spoilage pathogens in food and feed crops during refrigerated distribution and storage.1 These fungi can produce a variety of secondary metabolites known as Alternaria mycotoxins, such as tenuazonic acid, 1; alternariol, 2; alternariol methyl ether, 3; altenuene, 4; and tentoxin, 5 (Figure 1). These Alternaria mycotoxins are considered among the most common food contaminants, including in grains (such as wheat,2−6 sorghum,7 and corn8), vegetables (tomatoes9−13 and carrots14), as well as fruits (pomegranates,15 berries,16−18 citrus fruits,19 and apples20). Potential adverse health effects of the main Alternaria mycotoxins have been observed in both in vivo and in vitro studies. 1 is known to pose acute toxicity and induce embryonal death in microgram doses21 and cause severe dysplasia in mice.22 4 shows acute toxicity even more potent than 1.23 2 and 3 exert genotoxic and mutagenic effects on cultured mammalian cells.24 There are few toxicological data on 5 in the current literature. Despite their potential risks and widespread occurrence, to date, no specific global regulations governing these toxins in food have been set, which is probably due to the lack of sufficient toxicity and exposure data. However, in recent years, human dietary exposure to Alternaria mycotoxins has attracted the attention of the scientific community. Hickert et al.25 estimated the mean daily exposure to Alternaria mycotoxins in Germany using analytical results from 96 samples. In 2016, the European Food Safety Authority (EFSA) released a dietary exposure assessment for four individual Alternaria mycotoxins (i.e., 1−3 and 5) for the European population based on 4249 analyzed samples.26 This assessment recommended further © 2018 American Chemical Society
efforts to develop more sensitive methods to reduce the uncertainty associated with the exposure and generate more analytical data on Alternaria mycotoxins in relevant food commodities such as fruit and fruit products, and the report suggested that information available on the occurrences of Alternaria mycotoxins in fruit and fruit products is still limited. Sweet cherry is one of the most popular fruits worldwide because of its excellent sensory properties and high contents of nutrients and bioactive components. Precise data on the consumption of sweet cherry worldwide are not available, but based on the annual production of sweet cherry, the amount is expected to be high. According to the statistical data reported by the Food and Agriculture Organization (FAO) of the United Nations, 37531 tonnes of sweet cherry were produced in China in 2016,27 and the amount of production areas for sweet cherry increased almost linearly over 10 years.28 The consumption of sweet cherries in China in 2016 was estimated to be approximately 340000 tons, which accounts for 11% of the global consumption.29 Unfortunately, this fruit is susceptible to black leaf spot disease caused by microbial pathogens such as Alternaria spp., which has been isolated from the leaves of cherry trees in both China30 and Europe.31 The presence of Alternaria spp. in harvested sweet cherry fruits has also been documented,32−34 which means mycotoxins produced by Alternaria spp. may co-occur in sweet cherries and even in cherry-based products. Nevertheless, the occurrence of Alternaria mycotoxins in sweet cherries as well as their products remains unknown due to the lack of relevant analytical methods. Received: Revised: Accepted: Published: 11846
September 17, 2018 October 15, 2018 October 17, 2018 October 17, 2018 DOI: 10.1021/acs.jafc.8b05065 J. Agric. Food Chem. 2018, 66, 11846−11853
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of the Alternaria mycotoxins: (1) tenuazonic acid, (2) alternariol, (3) alternariol monomethyl ether, (4) altenuene, (5) tentoxin, (6) (ethyl-13C2)-tenuazonic acid, and (7) 2H3-tentoxin. an in-house Milli-Q Ultrapure water system (Millipore, Bedford, MA). Formic acid (99.0%), ammonium hydroxide solution (28−30% NH3), and ammonium bicarbonate (>99.9%) were obtained from Sigma-Aldrich. Sodium chloride (99.5%) was purchased from Sinopharm Chemical Reagent (Shanghai, China). Oasis HLB cartridges were obtained from Waters (Milford, MA). Sample Collection. Fifty-five fresh sweet cherry samples were randomly collected from supermarkets and farmers’ markets in Beijing. The samples varied in color from almost black to yellow, and 26 samples came from the main cherry-producing regions of China, including Beijing, Shandong, Liaoning, and Hebei provinces, 24 samples came from the USA, and 5 samples came from Canada. Twentyeight processed sweet cherry products, including 13 cherry jams, 12 dried cherries, and 3 canned cherries, were purchased from online stores, and the major brands were represented. Fourteen of the 28 sweet cherry products were imported from Germany (n = 3), France (n = 2), America (n = 3), Britain, Italy, Austria, Poland, Czech, and Denmark, and the remaining were domestically produced. The fresh samples were cleaned with ultrapure water and homogenized. Dried cherries were cut into small pieces with a knife, mixed with a 2-fold excess of water by weight, allowed to soak, and mashed. Cherry jams were mixed with an equal weight of water and mashed. All samples were stored at −20 °C until the next pretreatment step. Sample Preparation. Approximately 2.0 g of homogenized sample was weighed into a 15 mL centrifuge tube fortified with 6 and 7 at concentrations of 0.50 μg/kg and 0.05 μg/kg, respectively. After it was vortexed and placed in the dark for 30 min, the sample was extracted with 6 mL of acetonitrile by ultrasonication for 10 min, mixed with 2.0 g of sodium chloride, and vortexed for 30 s. The mixture was then centrifuged at 9000 rpm for 5 min at 4 °C. The supernatant (acetonitrile) was transferred into a glass vial and evaporated to dryness using a rotary evaporator at 120 rpm and 40 °C. The residue was reconstituted with 1 mL of methanol and 9 mL of water for SPE via an HLB cartridge preconditioned sequentially with 5 mL of methanol and 5 mL of water. After the sample was loaded, the cartridge was washed sequentially with 5 mL of methanol/water (40:60, v/v), 5 mL of methanol/water (40:60, v/v) containing 1% formic acid, and 5 mL of water. The cartridge was then dried under vacuum and eluted with 5 mL of methanol/acetonitrile (50:50, v/v) containing 1% ammonium hydroxide. The eluate was dried under a gentle stream of nitrogen at 40 °C. The residue was dissolved in 100 μL of methanol, vortexed with 900 μL of water, and then centrifuged at 14000 rpm for 5 min for UPLC-MS/MS analysis. UPLC-MS/MS Conditions. Chromatographic separations of Alternaria mycotoxins were performed on an LC-30A Ultra-Performance LC system (Shimadzu, Kyoto, Japan) equipped with a 100 mm × 2.1 mm i.d., 1.8 μm, HSS C18 column (Waters, Milford, MA). Methanol (solvent A) and 0.15 mmol/L ammonium carbonate in water (solvent B) were used as mobile phases at a flow rate of 0.30 mL/min. The initial mobile phase contained 20% A and was held for 1 min, and then the proportion of A was linearly increased to 100% over 3 min and then held for 1.5 min; finally, the column was returned to initial conditions and equilibrated for 1.5 min before the next injection.
Liquid chromatography coupled with a tandem mass spectrometry detector is commonly used for the determination of Alternaria mycotoxins in grains, vegetables, fruits, and their products (i.e., liquor, juice, jam, and puree).17,18,35−40 Antibodybased enzyme immunoassays have also been applied.41,42 However, because the specific, monoclonal antibodies are suitable only for one kind of Alternaria mycotoxin, multiple monoclonal antibodies are required to evaluate multiple substances. In addition, polyclonal antibodies suitable for multiple Alternaria mycotoxins are difficult to find. The biggest problem with the currently established methods is that it is not sufficiently sensitive, resulting in a high proportion of left-censored data (below the LOQs or/and LODs) in the analytical results,26 which in turn affects the accuracy of the evaluation. With the rapid advances in mass spectrometers, the sensitivity of the newest generation is several orders of magnitude better than that of the older generations. By optimizing the pretreatment conditions in conjunction with the newest generations of mass spectrometers, the sensitivity of the method would be greatly improved. To assess the level of Alternaria mycotoxins intake through cherries, both their levels in cherries and the consumption of cherries must be determined; however, there is little consumption data available for cherries and cherry-based products. Food frequency surveys can more accurately investigate food consumption data.43,44 In this study, we aimed to develop an accurate and sensitive method based on LC-ESI-MS/MS for simultaneous determination of five Alternaria mycotoxins in fresh sweet cherries, cherry jams, dried cherries, and canned cherries. The consumption data were collected by a web-based questionnaire. Then, exposure doses to Alternaria mycotoxins via fresh sweet cherry intake in the Chinese population were estimated preliminarily.
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MATERIALS AND METHODS
Chemicals and Reagents. 1 (>98.0%) and (acetyl-13C2)tenuazonic acid (6) (>99.0%, 50 μg/mL in acetonitrile) were purchased from Sigma-Aldrich (Saint Louis, MO). 2 (>98.0%), 3 (>99.0%), 4 (>99.0%), 5 (>99.2%), and 2H3-tentoxin (7) were obtained from Toronto Research Chemicals (Toronto, Canada). The individual stock solutions of 1−5 and 7 were prepared in methanol at 1000 μg/mL and stored in amber vessels at −20 °C. The working standard solutions at various concentrations were freshly prepared before use by combining aliquots of the stock solutions with a methanol/water (9:1, v/v) solution. HPLC-grade methanol and acetonitrile for sample extraction were from Dikma Technologies Inc. (Lake Forest, CA). LC-MS-grade methanol for the mobile phase was purchased from Honeywell International Inc. (Seelze, Germany). Ultrapure water was obtained using 11847
DOI: 10.1021/acs.jafc.8b05065 J. Agric. Food Chem. 2018, 66, 11846−11853
Article
Journal of Agricultural and Food Chemistry The column temperature was set at 40 °C, and the injection volume was 5 μL. The LC eluate was monitored by an LCMS-8060 triple quadruple mass spectrometer (Shimadzu, Japan) with an ESI interface operated in the negative mode. Multiple reaction monitoring (MRM) was employed. The interface voltage was 1.5 kV. Nitrogen gas (purity = 99.9%) was used as the nebulizing gas and drying gas at flow rates of 120 L/h and 300 L/h, respectively. The interface temperature, desolvation line temperature, and heat block temperature were set at 300, 250, and 400 °C, respectively. The parent and fragment ions for each toxin were determined based on the optimal response in a spiked sample. LabSolutions Ver.5.85 software was used for data acquisition and analysis. Method Validation. The accuracy and repeatability of the method, expressed as the recovery and relative standard deviation (RSD), respectively, were determined by analyzing blank samples (fresh cherry as well as related products) spiked with four different concentrations of the analyte in six replicates. Reproducibility was evaluated by performing analyses of the blank samples spiked with a medium concentration of the analyte on five consecutive days. The sensitivity of the method was evaluated by the limit of detection (LOD) and LOQ, which were the lowest spiked concentrations that could yield signal-to-noise ratios (S/N values) greater than 3 and 10, respectively. Matrix effects were calculated by the following equation.45 matrix effect (%) = [1 − (B/ A)] × 100%
(1)
where A represents the peak area of the standard solution and B represents the peak area of the matrix-matched standard solution at the same concentration. Matrix-matched calibration curves were conducted in all four types of samples (fresh cheery, cherry jam, dried cherry, and canned cherry) at a series of concentrations (1, 4: 0.02, 0.04, 0.1, 0.2, 0.4, 1.0, and 2.0 ng; 5: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 ng; 2: 0.06, 0.12, 0.3, 0.6, 1.2, 3.0, and 6.0 ng; 3: 0.003, 0.006, 0.015, 0.03, 0.06, 0.15, and 0.3 ng; 7: 0.05 ng; and 6: 0.1 ng). Internal standard calibration curves for 1 and 5 were obtained by performing a linear regression analysis on the ratio of the areas of the standard solution areas to the areas of the internal standards versus concentration. The matrix effects were observed to cause significant differences between different matrices. For highly contaminated samples, the extracts were diluted with ultrapure water to keep the level of analytes within the linear range of the method to ensure accurate quantitation. Dietary Questionnaire Survey and Exposure Assessment. Dietary Questionnaire Survey. A web-based food frequency questionnaire survey was conducted on May 14−21, 2018, to acquire consumption data regarding cherries and their products. The food frequency questionnaire was developed using the online platform provided by wenjuan.com and was published on WeChat through a Web site link (https://www.wenjuan.com/s/7J3iyu3/). The survey was anonymous, voluntary, and had no monetary incentive offered. The questionnaire mainly involved four topics: (1) basic information (sex, age, and weight); (2) whether the individual consumed cherries and their products between July and August of each year (persons who answered no were not asked additional questions); (3) the consumption frequency of cherries throughout the month; and (4) the amount of cherry product consumed per sitting. A total of 501 responses from participants were received. Additionally, 33 respondents were removed because of incomplete or illogical data. The remaining participants (305 women and 163 men) were categorized into four age groups: 15−24 years (n = 58), 25−34 years (n = 176), 35−44 years (n = 141), and 45 years or older (n = 93). Dietary Exposure Assessment. Lower, medium, and upper bounds were determined based on the contamination data obtained in this study. In the lower bound scenario, all nondetects were set to 0 μg/kg, while in the medium and upper bound scenario, the nondetects were set at half of the LOD and LOD, respectively. In addition, values below the LOQ were set at the LOD, at half of the LOQ, and at the LOQ.45 The food intake was expressed as grams per kilogram of
Figure 2. MRM chromatogram of the five Alternaria mycotoxin standards under the same condition using different reversed-phase columns: (A) BEH C8 column; (B) BEH C18 column; (C) COS C18 column; (D) HSS T3 column; and (E) HSS C18 column. 1, tenuazonic acid; 2, alternariol; 3, alternariol methyl ether; 4, altenuene; and 5, tentoxin. 11848
DOI: 10.1021/acs.jafc.8b05065 J. Agric. Food Chem. 2018, 66, 11846−11853
Article
Journal of Agricultural and Food Chemistry Table 1. Recoveries of the Five Alternaria Mycotoxins in Spiked Cherries and Cherry-Based Products fresh cherry spiked level (μg/kg) a
1
2a
3a
4a
5a
0.02 0.04 0.10 1.60 0.06 0.12 0.30 4.80 0.003 0.006 0.015 0.240 0.02 0.04 0.10 1.60 0.01 0.02 0.05 0.80
canned cherry
cherry jam
dried cherry
rec. (%)b
RSD (%)c
rec. (%)
RSD (%)
rec. (%)
RSD (%)
rec. (%)
RSD (%)
85.5 82.3 80.4 86.0 90.8 94.5 95.4 95.3 84.2 81.3 79.3 87.5 100.7 104.5 97.4 105.9 95.5 96.2 95.7 96.6
12.9 8.5 3.2 3.2 6.9 12.3 9.3 4.2 8.2 6.7 9.8 4.0 5.3 10.5 4.2 3.8 7.1 4.0 6.9 5.3
75.8 76.6 76.2 83.9 78.0 78.4 72.7 79.2 80.1 84.9 89.5 90.0 94.1 90.4 101.3 91.8 92.8 92.7 99.5 97.5
2.9 6.0 7.7 4.7 6.1 8.0 1.7 5.3 7.7 7.1 2.2 4.3 6.4 3.1 2.9 3.3 8.3 2.4 3.9 6.9
86.2 83.3 84.5 83.9 98.7 101.6 105.8 103.2 91.6 94.6 95.1 97.6 89.3 94.5 95.1 87.2 88.8 93.2 92.3 81.4
11.0 8.8 8.9 4.5 5.8 6.9 13.9 7.0 1.9 6.9 12.4 8.4 5.5 9.0 5.2 3.6 4.2 7.9 3.7 2.3
88.4 71.4 82.1 89.4 99.6 105.4 95.2 87.1 91.8 99.7 97.1 98.1 84.8 86.2 86.9 91.7 89.0 86.2 86.9 88.7
9.9 3.7 1.9 8.6 7.2 2.1 3.1 4.9 9.8 1.8 4.5 3.5 9.0 5.8 2.7 4.6 4.9 2.6 3.4 4.2
a 1, tenuazonic acid; 2, alternariol; 3, alternariol methyl ether; 4, altenuene; and 5, tentoxin. brec.: recovery, n = 6. cRSD: relative standard deviation, n = 6.
body weight (b.w.) per day and was evaluated by the following equation: food intake (g/kg b.w./day) = A × B/C
phenomenon remained. The CORTECS C18 and HSS T3 columns were adopted in the previous studies9,36 for the analysis of Alternaria mycotoxins. However, due to the end-capping and bigger surface area of the HSS C18 column, it afforded better retention and separation for all analytes as compared to the CORTECS C18 column and HSS T3 column in the present study. Since the pH tolerance of the HSS C18 column (pH 2−8) was much narrower than that of the BEH C18 column (pH 2−12), we varied the additive concentration from 0.5 to 0.15 mmol/L to decrease the alkalinity of the mobile phase. Optimization of the Sample Pretreatment Method. Sample Extraction. The extraction volume was optimized by comparing the recovery efficiency. For this, blank sweet cherry samples were spiked with five of the toxins. After evaporation, all samples were redissolved in 100 μL of methanol, vortexed with 900 μL of water, and then subjected to centrifugation prior to UPLC-MS/MS analysis. The recoveries of five Alternaria mycotoxins extracted with 6 mL of acetonitrile were 84.4−94.7%. Sample Purification. After sample extraction, an additional purification procedure was performed using SPE to reduce the interferences. Considering that the polarities of the target compounds vary substantially (logP from 0.87−3.32), Oasis HLB was selected because of its proven versatility and efficiency in the extraction of both polar and nonpolar compounds. The loading, washing, and elution solvents for the HLB column were optimized using mixtures of different proportions of methanol, water, formic acid, and ammonium hydroxide. Methanol/water (40:60, v/v) was used as the loading solvent to ensure that all of the analytes were retained on the cartridge without any loss. Five milliliters of methanol/ water (40:60, v/v) containing 1% formic acid was used to remove neutral and basic interferences, such as anthocyanidins and sugars, which were more polar than the target compounds. The addition of formic acid to the methanol/water (40:60, v/v) solution enhanced the hydrophobic interactions between 1 and the sorbent, and no analytes eluted during the wash procedure.
(2)
where A represents the frequency of cherry consumption, B represents the amount of cherries consumed per sitting, and C represents the body weight of the respondent. Dietary exposure to toxins was assessed based on a combination of the fixed toxin concentrations with the lower, medium, and upper bound means and maximum concentration multiplied by the percentiles (maximum, P99, P95, P90, P75, medium, mean, and minimum) of the consumption data. Data Analysis. Calculations and further data processing were performed using IBM SPSS Statistics 21. Due to the severely skewed distribution of the consumption data, a Kruskal−Wallis H test was employed to examine the differences between sex and age groups. A Mann−Whitney U test was used for analysis of the difference in toxin concentration and species between sweet cherries and cherry products.
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RESULTS AND DISCUSSION Optimization of UPLC Conditions. The ideal separation situation is the obtention of a narrow and sharp peak and good separation with no additives in the mobile phase. However, because of its acidity, 1 will not elute with a suitable peak shape without an additive in the mobile phase. The use of ammonium bicarbonate as a mobile phase additive for the separation of Alternaria mycotoxins has been reported previously.11,39 In our preliminary experiments, a BEH C18 column was used to separate the five analytes. Although methanol/water with an additive improved the peak shapes slightly, satisfactory peak shapes and retention times could not be achieved for 1 because of its polarity. Thus, reversed-phase chromatography columns other than BEH C18, including BEH C8, HSS T3, HSS C18, and CORTECS C18 columns, were tested and compared. As shown in Figure 2, the retention of 1 on the BEH C18 column was weak and the peak tailed. The same results can be found in the chromatograms of Zhao et al.11 and Wang et al.36 For the BEH C8 column, the retention of 1 was improved, but the tailing 11849
DOI: 10.1021/acs.jafc.8b05065 J. Agric. Food Chem. 2018, 66, 11846−11853
Article
11850
5.7 2.8 4.7 2.3 1.1 0.018 0.066 0.003 0.023 0.008 0.005 0.020 0.001 0.007 0.002 7.0 10.1 7.0 10.4 6.5 5.6 7.5 5.1 6.3 4.7 0.018 0.038 0.002 0.020 0.007 a
1, tenuazonic acid; 2, alternariol; 3, alternariol methyl ether; 4, altenuene; and 5, tentoxin. bn = 6.
0.005 0.011 0.001 0.006 0.002 4.2 3.6 5.1 7.2 4.8 2.3 3.0 2.0 4.2 0.8 0.020 0.042 0.002 0.014 0.006 0.007 0.013 0.001 0.004 0.002 5.6 4.8 4.9 7.2 5.7 0.016 0.055 0.002 0.019 0.007 0.005 0.017 0.001 0.006 0.002 1 2 3 4 5
4.6 3.4 1.6 2.8 1.4
intraday precision (%) intraday precision (%) intraday precision (%) LOD interday precision (%)b intraday precision (%)b LOQ LOD toxinsa
fresh cherry
Table 2. LOD, LOQ, and Precision of the Method
LOQ
canned cherry
interday precision (%)
LOD
LOQ
cherry jam
interday precision (%)
LOD
LOQ
dried cherry
interday precision (%)
More than 70% of the 2−5 eluted from the HLB column with 5 mL of methanol/acetonitrile (50:50, v/v), while only 50% of 1 was recovered. Therefore, 1% ammonium hydroxide was added to the elution solvent, and the recovery of 1 increased to 80%. The matrix effect was evaluated for the proposed method. The results revealed that signal suppression of 5, 4, and 1 in cherry and cherry products was greatly reduced by the use of HLB, leading to matrix effects of no more than 30%. However, 2 and 3, with relatively strong retention capabilities, still displayed severe ion suppression (>70%) effects, which was similar to the QuEChERs method developed for Alternaria mycotoxins in fruit and vegetable juices by Jeroen et al.,4 in which signal suppression greater than 75% was observed for 3 and 2. In another study,8 the SPE method presented matrix suppression that exceeded 80%. Method Performance. Good linearity was obtained for all analytes with correlation coefficients (R2) of the matrixmatched calibration curves all above 0.99. The mean recoveries of the target compounds ranged from 79.3−104.5% in fresh sweet cherries, 83.3−105.8% in cherry jams, 71.4−105.4% in dried cherries, and 72.7−101.3% in canned cherries with RSDs at each fortification level for each compound
