Natural Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods Marketed in South Korea by LC–MS/MS

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity (R2 > 0.999), the limit of detection (0.04–1.67 μg/kg), the limit of quantification (0.12–5.06 μg/kg), recovery (80.0–114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 μg/kg for AOH, 4.36 μg/kg for AME, 0.14 μg/kg for ALT, 0.11 μg/kg for ATX-I, 0.43 μg/kg for TEN, and 104.56 μg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.


. Recovery and Precision
The Alternaria toxins were classified into three different groups based on the similarity of their reported occurrence levels. For validation, the rice, sesame, tomato, and apple juice samples were spiked with 5, 10, and 20 μg/kg of AOH, AME, ALT, and ATX-I, 25,

. Recovery and Precision
The Alternaria toxins were classified into three different groups based on the similarity of their reported occurrence levels. For validation, the rice, sesame, tomato, and apple juice samples were spiked with 5, 10, and 20 µg/kg of AOH, AME, ALT, and ATX-I, 25, 50, 100 µg/kg of TEN, and 50, 100, 200 µg/kg of TeA. Each value was rounded to the first decimal place. The intra-and inter-day validation results are listed in Table 2. The mean inter-and intra-recovery ranges of the six analytes in rice, sesame seed, tomato, and apple juice were 82.1-114.7%, 80.0-94.9%, 82.7-110.7%, and 85.9-114.3% ranges, respectively. The mean relative standard deviation (RSD), an estimate for the precision, of the six analytes ranged from 4.3 to 15.6% in rice, 2.5 to 17.7% in sesame, 3.2 to 15.1% in tomato, and 4.1 to 15.4% in apple juice. All of the estimated recovery and precision values were within an acceptable range according to the AOAC Official Methods of Analysis (2016) Guidelines for Standard Method Performance Requirements. Specifically, the guideline recommend that the recovery values should be within 60-115% and 80-110% for concentrations of 10 and 100 µg/kg, respectively [25]. Moreover, the RSD values recommended by the AOAC for concentrations of 10 and 100 µg/kg are <21% and <15%, respectively.

Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods
A total of 152 marketed food samples, including cereal grains (n = 31), pulses (n = 15), seasoning foods (n = 16), nuts and seeds (n = 22), beverages (n = 32), vegetables (n = 16), and fruits (n = 20) were analyzed using the validated method. The obtained contamination levels of Alternaria toxins are reported in Figure 2 and Table 3, which include the incidence, mean concentrations, and concentration ranges of the six Alternaria toxins in each food product. The mean inter-and intra-recovery ranges of the six analytes in rice, sesame seed, tomato, and apple juice were 82.1-114.7%, 80.0-94.9%, 82.7-110.7%, and 85.9-114.3% ranges, respectively. The mean relative standard deviation (RSD), an estimate for the precision, of the six analytes ranged from 4.3 to 15.6% in rice, 2.5 to 17.7% in sesame, 3.2 to 15.1% in tomato, and 4.1 to 15.4% in apple juice. All of the estimated recovery and precision values were within an acceptable range according to the AOAC Official Methods of Analysis (2016) Guidelines for Standard Method Performance Requirements. Specifically, the guideline recommend that the recovery values should be within 60-115% and 80-110% for concentrations of 10 and 100 μg/kg, respectively [25]. Moreover, the RSD values recommended by the AOAC for concentrations of 10 and 100 μg/kg are < 21% and < 15%, respectively.

Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods
A total of 152 marketed food samples, including cereal grains (n = 31), pulses (n = 15), seasoning foods (n = 16), nuts and seeds (n = 22), beverages (n = 32), vegetables (n = 16), and fruits (n = 20) were analyzed using the validated method. The obtained contamination levels of Alternaria toxins are reported in Figure 2 and Table 3, which include the incidence, mean concentrations, and concentration ranges of the six Alternaria toxins in each food product.   Among the dibenzo-α-pyrone derivatives, the highest incidence was found for AOH (27/152), followed by AME (25/152) and ALT (5/152). The mean concentration of AOH in all of the tested samples was 2.77 µg/kg, with levels ranging from 0.66 to 105.49 µg/kg. The food product with the highest occurrence of AOH was buckwheat, which was also the food product with the highest incidence (mean = 26.68 µg/kg, maximum = 105.49 µg/kg, incidence = 80%). The mean concentration of AME in all of the tested samples was 4.36 µg/kg, with a range of 0.20 to 310.82 µg/kg, which was slightly wider than that of AOH. The highest concentration of AME was found in black sesame (mean = 106.49 µg/kg, maximum = 310.82 µg/kg, incidence = 80%), and the food product with the highest AOH detection frequency was buckwheat (mean = 6.18 µg/kg, maximum = 21.89 µg/kg, incidence = 100%). ALT was detected in five samples of the nuts and seeds group, whereas none of the tested samples of cereal grains, pulses, seasoning foods, beverages, vegetables, and fruits was contaminated with ALT. The mean occurrence level of ALT was 0.14 µg/kg, with levels ranging from 1.13 to 10.71 µg/kg. The highest level of ALT was determined in black sesame (mean = 3.95 µg/kg, maximum = 10.71 µg/kg, incidence = 80%). Interestingly, all of the detected ALT in the examined food samples co-occurred with at least two other Alternaria toxins (Figure 3). Among the dibenzo-α-pyrone derivatives, the highest incidence was found for AOH (27/152), followed by AME (25/152) and ALT (5/152). The mean concentration of AOH in all of the tested samples was 2.77 μg/kg, with levels ranging from 0.66 to 105.49 μg/kg. The food product with the highest occurrence of AOH was buckwheat, which was also the food product with the highest incidence (mean = 26.68 μg/kg, maximum = 105.49 μg/kg, incidence = 80%). The mean concentration of AME in all of the tested samples was 4.36 μg/kg, with a range of 0.20 to 310.82 μg/kg, which was slightly wider than that of AOH. The highest concentration of AME was found in black sesame (mean = 106.49 μg/kg, maximum = 310.82 μg/kg, incidence = 80%), and the food product with the highest AOH detection frequency was buckwheat (mean = 6.18 μg/kg, maximum = 21.89 μg/kg, incidence = 100%). ALT was detected in five samples of the nuts and seeds group, whereas none of the tested samples of cereal grains, pulses, seasoning foods, beverages, vegetables, and fruits was contaminated with ALT. The mean occurrence level of ALT was 0.14 μg/kg, with levels ranging from 1.13 to 10.71 μg/kg. The highest level of ALT was determined in black sesame (mean = 3.95 μg/kg, maximum = 10.71 μg/kg, incidence = 80%). Interestingly, all of the detected ALT in the examined food samples co-occurred with at least two other Alternaria toxins (Figure 3). ATX-I, a perylene quinone derivative, was detected in four samples of cereal grains, one sample of nuts and seeds, and two samples of beverages. ATX-I was not found in any of the examined samples of pulses, seasoning foods, vegetables, and fruits. The mean concentration of ATX-I in all monitored foods was 0.11 μg/kg, and its levels ranged from 0.50 to 7.81 μg/kg; this was the lowest contamination level among the six Alternaria toxins. The highest contamination level and incidence of ATX were found in buckwheat (mean = 2.17 μg/kg, maximum = 7.81 μg/kg, incidence = 40%).
TEN, a cyclic tetrapeptide, was found in all monitored samples at levels ranging from 2.77 to 14.64 μg/kg. The average TEN contamination level found in the 152 tested samples was 0.43 μg/kg, which was similar to that of ALT and ATX-I. Only nine samples were positive for TEN, whose highest concentration and incidence were observed in sorghum ATX-I, a perylene quinone derivative, was detected in four samples of cereal grains, one sample of nuts and seeds, and two samples of beverages. ATX-I was not found in any of the examined samples of pulses, seasoning foods, vegetables, and fruits. The mean concentration of ATX-I in all monitored foods was 0.11 µg/kg, and its levels ranged from 0.50 to 7.81 µg/kg; this was the lowest contamination level among the six Alternaria toxins. The highest contamination level and incidence of ATX were found in buckwheat (mean = 2.17 µg/kg, maximum = 7.81 µg/kg, incidence = 40%).
TEN, a cyclic tetrapeptide, was found in all monitored samples at levels ranging from 2.77 to 14.64 µg/kg. The average TEN contamination level found in the 152 tested samples was 0.43 µg/kg, which was similar to that of ALT and ATX-I. Only nine samples were positive for TEN, whose highest concentration and incidence were observed in sorghum (mean = 3.48 µg/kg, maximum = 14.64 µg/kg, incidence = 40%) and buckwheat (mean = 5.75 µg/kg, maximum = 13.00 µg/kg, incidence = 60%). All ALT-positive samples were co-contaminated with other Alternaria toxins (Figure 3). The levels of ATX-I, ALT, and TEN in marketed foods from South Korea were similar to or lower than those recently reported by Xing  TeA, which is classified as a tetramic acid, was a major contaminant in this study. Among the 152 monitored samples, 39.5% (60/152) were contaminated with TeA at concentrations ranging from 1.26 to 4028.18 µg/kg, with a mean value of 104.56 µg/kg. TeA was detected in all of the 16 examined seasoning foods, and hot pepper powder was a major contributor to the TeA contamination (mean = 1845.95 µg/kg, maximum = 4028.18 µg/kg, incidence = 100%). Furthermore, the five food products with the highest concentration of TeA were also all red pepper powder products (1104.98-4028.18 µg/kg). This represents a lower occurrence level than those previously reported by Gambacor-ta et al. (2019) [28] (mean concentration in red chili = 27,255.5 µg/kg) and Mujahid et al. (2020) [29] (maximum concentration in chili items = 20,478 µg/kg). The incidence of TeA was high in the following order: black sesame (mean = 335.66 µg/kg, maximum = 911.63 µg/kg, incidence = 100%), tomato puree (mean = 211.07 µg/kg, maximum = 882.22 µg/kg, incidence = 100%), perilla seed (mean = 138.64 µg/kg, maximum = 476.19 µg/kg, incidence = 100%), and tomato ketchup (mean = 73.26 µg/kg, maximum = 91.10 µg/kg, incidence = 100%). Among the seasoning foods investigated, the contamination levels of TeA in tomato products (ketchup and puree) were similar to or lower than those reported in previous studies [10,22,30,31].

Correlation between Naturally Occurring Alternaria Toxins
The correlations between naturally co-occurring Alternaria toxins were investigated in cereal grain, seasoning food, and nuts and seeds, representing the three most frequently contaminated food categories (Figure 4). The correlation between Alternaria toxins was visualized using the 'corrplot' R package (version 4.1.2). ALT was not detected in any of the cereal grain samples (n = 31), and a positive correlation was observed between the contamination levels of all detected Alternaria toxins. The Spearman's rank correlation coefficients (rs) were 0.95 (AOH and AME), 0.89 (AOH and ATX-I), 0.82 (AME and TEN), 0.81 (AME and ATX-I), 0.75 (TEN and TeA), 0.67 (AOH and TEN), 0.62 (ATX-I and TEN), 0.45 (AME and TeA), 0.21 (AOH and TeA), and 0.06 (ATX-I and TeA). These results indicated that the wheat and wheat-based products examined herein showed a similar trend to those reported by Zhao et al. (2015) and Xu et al. (2016) [16,17]. ALT was not detected in any of the cereal grain samples (n = 31), and a positive correlation was observed between the contamination levels of all detected Alternaria toxins. The Spearman's rank correlation coefficients (r s ) were 0.95 (AOH and AME), 0.89 (AOH and ATX-I), 0.82 (AME and TEN), 0.81 (AME and ATX-I), 0.75 (TEN and TeA), 0.67 (AOH and TEN), 0.62 (ATX-I and TEN), 0.45 (AME and TeA), 0.21 (AOH and TeA), and 0.06 (ATX-I and TeA). These results indicated that the wheat and wheat-based products examined herein showed a similar trend to those reported by Zhao et al. (2015) and Xu et al. (2016) [16,17].
Only three types of Alternaria toxins, AOH, AME, and TeA, were detected in seasoning foods. In this food group, TeA co-occurred with AOH and/or AME in 56.3% of the samples, and a particularly high contamination level of TeA was observed. However, only a weak positive correlation was observed between the detected toxins (r s = 0.42 for AOH and AME, 0.38 for AOH and TeA, and 0.34 for AME and TeA).
All six Alternaria toxins were detected in the nuts and seeds group. A high positive correlation was observed between the dibenzo-α-pyrone derivatives (r s ≥ 0.94). Moreover, a positive correlation was observed between all Alternaria toxins except for TEN, and the r s value between the toxins showing a positive correlation was higher than 0.76. Interestingly, TEN showed a weak negative correlation with all other Alternaria toxins only in the nuts and seeds group. To the best of our knowledge, our study is the first to demonstrate that the contamination levels of Alternaria toxins may show a negative correlation depending on the food category. Particularly, the r s values were −0.16 (AME and TEN, ALT and TEN), −0.15 (AOH and TEN), and −0.09 (ATX-I and TEN, TeA and TEN).

Estimation of Daily Dietary Exposure to Alternaria Toxins
The estimated daily exposure of each ATs from marketed food in the South Korean population is summarized in Table 4. For the mean exposure scenario, the average daily exposure levels were 0.0118 (LB)-0.0506 ng/kg b.w./day (UB) for AOH, 0.0101 (LB)-0.0378 ng/kg b.w./day (UB) for AME, 0.0001 (LB)-0.0364 ng/kg b.w./day (UB) for ALT, 0.0022 (LB)-0.0381 ng/kg b.w./day (UB) for ATX-I, 0.0039 (LB)-0.1744 ng/kg b.w./day (UB) for TEN, and 3.7094 (LB)-3.8499 ng/kg b.w./day (UB) for TeA in all food samples. Seasoning foods were the main contributors for dietary exposure to AOH and TeA. Nuts and seeds were the major contributors for dietary exposure to AME and ALT. Beverages and cereal grains were the highest contributors for daily dietary exposure to ATX-I and TEN, respectively. For the extreme exposure (95th percentile) scenario, the average daily exposure levels were 0.0935 (LB)-0.2255 ng/kg b.w./day (UB) for AOH, 0.0733 (LB)-0.1572 ng/kg b.w./day (UB) for AME, 0.0003 (LB)-0.1534 ng/kg b.w./day (UB) for ALT, 0.0072 (LB)-0.1889 ng/kg b.w./day (UB) for ATX-I, 0.0591 (LB)-0.8437 ng/kg b.w./day (UB) for TEN, and 14.3004 (LB)-15.0344 ng/kg b.w./day (UB) for TeA in all food samples. Cereal grains were the most important contributors for dietary exposure to AOH, AME, ATX-I, and TEN estimated by extreme food intake scenario due to their high consumption. Nuts and seeds and seasoning foods were the highest contributors for daily dietary exposure to ALT and TeA, respectively.

Conclusions
Our study validated an LC-MS/MS-based method with SPE clean-up for the simultaneous analysis of six ATs in four different food matrices to investigate the natural occurrence of ATs in marketed foods in South Korea. Further, the co-occurrence of six ATs and correlation between different levels of toxin were investigated. Processed tomato products are among the foods that are susceptible to contamination by Alternaria toxins. In South Korea, the occurrence of toxins in processed tomato products (ketchup, puree, juice) was similar to those reported in other countries. TeA was the most frequently detected Alternaria toxin among all of the tested food categories, and the highest TeA level was found in hot pepper powder. Although the toxin occurrence levels were lower than those reported in previous studies, continuous monitoring is needed for risk management of red pepper powder, which is among the most widely consumed products in South Korea. Additionally, 44% of positive samples were co-contaminated with at least two Alternaria toxins. The overall estimated daily dietary exposures of South Koreans to ATs were lower than those reported in previous studies from other countries. Collectively, our results highlight the need for further monitoring and risk assessment of co-occurring Alternaria toxins in agricultural and food products.

Sample Collection
A total of 152 marketed agricultural products and processed food samples were purchased from online retailers, supermarkets, and local markets in 2020. The collected samples were classified into as followings: cereal grains (rice, brown rice, wheat flour, barley, sorghum, and buckwheat), pulses (soybean, kidney bean, and lentils), seasoning foods (hot pepper powder, tomato ketchup, and tomato puree), nuts and seeds (sesame, perilla seed, black sesame, and sunflower seed), beverages (apple juice, orange juice, grape juice, tomato juice, black tea, corn silk tea, barley tea, and soybean milk), vegetables (tomato, onion, and Korean cabbage), and fruits (apple, mandarin, grape, and watermelon). The samples were homogenized and stored at −20 • C, followed by equilibration at room temperature prior to analysis.

Sample Preparation
After purification of the Alternaria toxins from rice, sesame seed, apple juice, and tomato, toxin determination in tomato, wheat, and sunflower seeds was performed by SPE clean-up and HPLC-MS/MS, using the EN 17521-2021 standard method developed by the Slovenian Institute for Standardization (SIST), with some modifications [32]. Briefly, 2 g of sample was extracted with 15 mL of MeOH/water/acetic acid (85:14:1, v/v/v) by shaking for 45 min at 270 rpm, followed by centrifugation of the extract for 10 min at 3100× g. The supernatant (7.5 mL) was diluted with an equal volume of 1% acetic acid solution (v/v). The Alternaria toxins were cleaned up using solid-phase extraction with polymer-based hydrophilic-lipophilic balanced (HLB) SPE cartridges (200 mg, 6 mL) purchased from Waters (Milford, MA, USA). The cartridges were conditioned with 7 mL of MeOH and equilibrated with 7 mL of water and 4 mL of 1% acetic acid. After the equilibration solvent completely passed through the cartridges, their bottom was closed and 3 mL of 1% acetic acid was added. A syringe was attached to the cartridge to load the diluted supernatant and allow the cartridge to flow. Then, the cartridge was washed with 7 mL of 2%