| Objective To develop a simultaneous analytical method for Alternaria mycotoxins detection in foods and conduct a survey on the natural occurrence of tenuazonic acid (TeA), alternariol (AOH), alternariol monometyl ether (AME) and tentoxin (TEN) in wheat flour and wheat-based foods, fresh tomato and tomato-based foods as well as fresh citrus and citrus-based foods. Thein vitro digest modelsfor evaluati the bioaccessibilities of and the four Alternaria toxins were established. Meanwhile, the Caco-2 monolayer cell lines transfering model was established for evaluating the bioavailabilities of these four Alternaria toxins. The risks assessment of dietary exposure to the four Alternaria mycotoxins in Chinese populations were carried out based on the threshold of toxicological concern (TTC) method.Methods (1) An Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) collaborative analysis method was developed by optimization of both MS and chromatography conditions. (2) TeA, AOH, AME and TEN concentrations in 323 wheat flour and wheat flour based foods,70 tomato and tomato based foods,86 citrus and citrus based foods collected from Henan, Shandong, Anhui, Jilin and Beijing provinces were determined by the UPLC-MS/MS. (3) A in vitro digest model for evaluating the bioaccessibility of the four Alternaria toxins in wheat-based foods was developed by simulating the physiological digestion conditions of gastrointestinal tract and the optimal model parameters were investigated as well as model verification using sorbents of mycotoxins. A cell transportation model was developed using human colon carcinoma (Caco-2) cells for the transport of TeA, AOH and AME. Bioavailability rates together with the oral bioaccessibility of 3 Alternaria toxins were studied. (4) The Threshold of Toxicological Concern (TTC) methodology was employed for risk assessment of the human chronic dietary exposure to the four Alternaria mycotoxins of the overall population and different age groups and the internal exposure was primarily evaluated according to the results of bioavailability.Results (1) LODs of Alternaria toxins analyzed by UPLC-MS/MS method were between 0.1 μg/kg and 8 μg/kg. Recovery of the duplicate samples ranged from 58.9-116.0%, which met the requirement of sample analysis. Relative standard deviation (RSD) of replicability and intra-laboratory replicability ranged from 3.0-12.4%, all lower than 20%, which fulfilled the requirement of practice sample determination. (2) TeA was the main Alternaria toxins detected in food samples analyzed. The occurrence rate of TeA in wheat flour samples was 98.8% with levels ranging from 1.76 to 520 μg/kg (average=88.4 μg/kg). Occurrence of AOH, AME and TEN inwheat flour samples were 6.1%,91.2% and 76.8%, with contamination levels of 16.0-98.7 μg/kg (average=30.2 μg/kg),0.320-61.8 μg/kg (average=3.77 μg/kg) and 2.25-129 μg/kg (average=27.1 μg/kg), respectively. A regional difference in contamination levels of TeA were relevant to the sites of sampling with a descending order as follows, Henan, Anhui, Beijing, Shandong and Jilin. Positive correlations were found between the concentrations of AOH and AME (r=0.904, P<0.05), TeA and AME (r=0.757, P<0.01), TeA and AOH+AME (r=0.860, P<0.01), TEN and TeA (r=0.747,P<0.01) in wheat based foods. TeA was detected in all tomato ketchup and tomato juice samples with levels ranging from 10.2 to 1787 μg/kg (average=338 μg/kg) and from 7.4 to 278 μg/kg (average=87 μg/kg), respectively. Concentrations of TeA in imported tomato ketchup samples were significantly higher than those of domestic producers (P<0.05). Contamination levels of AOH and AME were from 2.5 to 300 μg/kg (average=80 μg/kg) and from 0.32 to 38 μg/kg (average=8.0 μg/kg), respectively with a range of 1.53-15.8 μg/kg (average=4.9 μg/kg) for TEN. AOH was not detected in tomato juice samples and AME was detected from 0.20 to 5.8 μg/kg (average=1.42 μg/kg). TeA was detected in 25% of the citrus juice samples with a range of 1.21-4.3 μg/kg(average=1.80 μg/kg), while AOH and TEN were not detected. (3) A digestion model in vitro for the study of bioaccessibility and bioavailability wasdeveloped. Oral bioaccessibility rates of TeA, AOH and AME were 72.1±5.4%, 7.0±0.8% and 10.2±1.0%, respectively. A Caco-2 model was successfully developed. Absorption rates of different concentrations and time intervals ranged from 1.5% to 63.0%. TeA was a mycotoxin that could be easily absorbed.(4)The average and 97.5th chronic dietary exposure of AOH and AME in the overall population and different age groups exceeded the relevant TTC values, among which dietary exposures of AOH and AME in the age group of 2-6 were higher than any other age group, followed by the age group of 7-17 and the internal UB exposure of AOH in overall population and different age groups with high consumption still exceeded the TTC value. Attention should be paid to its health risk. The average chronic dietary exposure of TeA and TEN in the overall population and different age groups were lower than the TTC value with much lower health risks.Conclusions The analysis method developed was sensitive with high specificity and met the anlysis requirement of the fou Alternaria toxins in different food matrix. The Chinese wheat and wheat based foods, tomato products and citrus products were contaminated by the four Alternaria toxins with high prevalence of TeA. The Caco-2 model developed was sensitive and stable and could be used for the study of bioavailability of the 3 Alternaria toxins. High potential health risk of AOH and AME to the population in China due to high dietary exposure through wheat flour and wheat flour products was found. More attention should be paid to their health risks. More chronic toxicological information should be obtained for further health risk assessment of AOH and AME.. |
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