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Article

Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS

by
Rastislav Boško
1,2,*,
Marek Pernica
2,
Sylvie Běláková
2,
Marie Bjelková
3 and
Helena Pluháčková
1
1
Department of Crop Science, Breeding and Plant Medicine, Faculty of AgriSciences, Mendel University in Brno, Zemědělská 1, CZ-61300 Brno, Czech Republic
2
Research Institute of Brewing and Malting, Mostecká 7, CZ-61400 Brno, Czech Republic
3
Agritec Plant Research, Zemědělská 2520/16, CZ-78701 Sumperk, Czech Republic
*
Author to whom correspondence should be addressed.
Toxins 2022, 14(4), 258; https://doi.org/10.3390/toxins14040258
Submission received: 7 March 2022 / Revised: 28 March 2022 / Accepted: 4 April 2022 / Published: 6 April 2022
(This article belongs to the Special Issue Reduction and Control of Mycotoxins along Entire Food and Feed Chain)
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Abstract

:
Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020–2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7–290.2 µg/kg and HT-2 toxin 157.0–319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8–69.9 µg/kg and HT-2 toxin was 24.2–75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.
Keywords:
milk thistle; T-2 toxin; HT-2 toxin; validation method; immunoaffinity column; UPLC-MS/MS
Key Contribution: T-2 and HT-2 toxins content in grown milk thistle harvested from a breeding station in the Czech Republic during 2020–2021 was studied. It was confirmed that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of mycotoxins in milk thistle seeds. All samples were contaminated with mycotoxins T-2 and HT-2. The use of milk thistle contaminated with hepatotoxic mycotoxins may reduce its health benefits and, in extreme cases, may be harmful to health.

1. Introduction

Milk thistle [Silybum marianum (L.) Gaertn.] is an annual, and sometimes biennial, medicinal plant of the family Asteraceae cultivated in agriculture, and it is commonly used as a medicinal drug [1,2]. In recent years, there has been a significant increase in demand from domestic and foreign processors, mainly from the pharmaceutical, cosmetics and feed industries. The milk thistle has a significant share in increasing the growing areas of medicinal plants and is the most cropped medicinal plant in the Czech Republic [1].
The subject of the crop are achenes—Silybi mariani fructus (Ph. Eur.)—and it has been used in health remedies since the time of ancient Greece [1,3]. The main beneficial constituent of milk thistle is silymarin—a complex of flavonolignans containing silybin A, silybin B, isosilybin A, isosilybin B, silydianin and silychristin—which exerts a positive effect on liver health [4,5,6]. Milk thistle also contains flavonoids—taxifolin and quercetin. Other potentially beneficial attributes of milk thistle include its antioxidant, antihypercholesterolemic and chemoprotective effects against lung and prostate cancer [7,8,9,10]. Milk thistle has anticancer activity, anti-inflammatory, immunomodulatory activity and neuroprotective potential [11].
The content of the silymarin complex depends on the variety of a milk thistle, e.g., the variety Silyb is approximately 2% and the minimum content according to the pharmacopoeia is 1% of silymarin complex per kilo of drug [1].
With the recent natural therapy revolution, the consumption of milk thistle supplements has widely spread among consumers in Europe [12]. Herbal medicine has been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. However, contaminants in herbal medicines produce severe problems that have seriously affected the value of herbal products and damaged human health [13].
As one of the major contaminants, mycotoxins are the secondary metabolites produced by various species of filamentous fungi, such as Aspergillus, Penicillium, Fusarium or Alternaria, and trigger several ailments of the kidneys, liver, skin and respiratory system. Among mycotoxins, aflatoxins, fumonisins, ochratoxin A, zearalenone and trichothecenes such as deoxynivalenol, T-2 toxin or HT-2 toxin are the most frequently detected mycotoxins in herbal medicines [14,15,16,17]. Generally, mycotoxins can develop either in the pre-harvest, post-harvest or storage stages. Climate change, poor storage and damage from harvest processing make crops more susceptible to mycotoxin contamination. In particular, milk thistle is characterized by the non-uniform ripening time of an individual flower head on the plant, which represents ideal conditions for the growth of fungi [15,16,17].
Mycotoxins have a different chemical structure, on which their metabolism and degree of toxicity in the organism depend. The genus Fusarium is one of the toxicogenic fungi with a high production of various mycotoxins. The optimal conditions for the growth and production of mycotoxins by microscopic fibrous fungi of the genus Fusarium are: temperature 15–25 °C, relative humidity 20–25%, water activity 0.98–0.99 and weakly acidic pH of the environment. The three most important groups of mycotoxins synthesized by the genus Fusarium are: trichothecenes, zearalenone and fumonisins [18,19].
The Fusarium toxins such as T-2 toxin, HT-2 toxin, diacetoxyscirpenol or neosolaniol are classified as type A trichothecenes [20]. Their main producers are Fusarium sporotrichioides, F. poae, F. langsethiae or F. acuinatum. As dominant field fungi in Europe are widely held in cereal as natural contaminants or other plants, e.g., medicinal plants including milk thistle [9,21,22,23,24].
The content of mycotoxins in milk thistle has been analysed in several studies. Fusarium mycotoxins such as T-2 and HT-2 toxins, zearalenone, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, neosolaniol or enniatins and beauvericins have been found in the samples, as well as high levels of Alternaria mycotoxins such as alternariol, alternariol-methyl ether, tentoxin or tenuazonic acid [14]. Fumonisins, aflatoxins, citrinin have been detected in several medicinal plants, including milk thistle [25]. Nivalenol, ochratoxin A, i.a., have been determined by Arroyo-Manzanares et al. in a multiclass mycotoxin analysis [26]. Fenclova [27] further analysed sterigmatocystin or mycophenolic acid.
Many analytical methods have been published concerning the determination of mycotoxins in different matrices. In general, chromatographic methods such as liquid chromatography coupled with mass spectrometry is the most commonly used technique for mycotoxin analysis, with other analytical techniques being seldom used due to their limited sensitivity and selectivity [28].
T-2 toxin, which belongs to the type A trichothecenes, has one of the highest toxicities of all the trichothecenes [29]. T-2 toxin is often produced by different Fusarium species which may grow on a variety of grains or seeds, especially in cold climate regions or during wet storage conditions [29,30]. In addition to fungal genetics, T-2 toxin production can be significantly influenced by environmental conditions, such as temperature, humidity and the type of substrate [31]. T-2 toxin is non-volatile and resistant to degradation in different environments, such as light and temperature, but it is effectively deactivated by strong acid or alkaline conditions. T-2 toxin causes a large range of toxic effects in animals, such as weight loss, decreases of blood cell and leukocyte count, reduction in plasma glucose and pathological changes in the liver and stomach. Furthermore, T-2 toxin is associated with an increased infection rate and induction of apoptosis [32,33,34,35,36,37,38]. T-2 toxin was considered as the cause of alimentary toxic aleukia (ATA) or Kashin–Beck disease [39]. Toxic effects of these mycotoxins are well documented in animals. In particular, T-2 toxin is a potent inhibitor of protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, both in vitro and in vivo, and has immunosuppressive and hematotoxic effects [39,40,41].
T-2 toxin is mainly metabolized into HT-2 toxin, a deacetylated form of T-2 toxin, which has comparable toxicity with T-2 toxin (Figure 1). A small amount of T-2 toxin can also be metabolized into diacetoxyscirpenol and/or neosolaniol [42,43,44,45,46]. The EFSA Panel on Contaminants in the Food Chain (CONTAM) established a tolerable daily intake (TDI) for T-2 toxin and HT-2 toxin of 0.02 μg/kg body weight per day for the sum of T-2 and HT-2 toxins [47].
The aim of this study was to develop and validate the analytical UPLC-MS/MS method using EASI-EXTRACT® T-2 & HT-2 immunoaffinity columns for the determination of T-2 and HT-2 toxins in milk thistle samples. The effect of row width (individual variants) was investigated (n = 4) to determine the effect of edge position in the plot of the randomized experiment. The effect of the climatic conditions of the year on the content of selected mycotoxins was also investigated.

2. Results

2.1. Method Validation

The analytical method was validated for selected parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy. Calibration curves of the selected mycotoxins were linear in the working range of LOQ–1000 ng/mL. The coefficients of determination (R2) were 0.9998 and 0.9999 for T-2 and HT-2 toxin, respectively; the regression equations were (y = 3298x − 1339.1) and (y = 1047.4x − 669.79) for T-2 and HT-2 toxins, respectively.
Detection and quantification limits were determined using consecutive dilution until the signal-to-noise ratio became unsuitable, and the concentrations were calculated as the ratio of S/N = 3 and S/N = 10 for LOD and LOQ, respectively. For T-2 toxin, LOD 0.04 μg/kg and LOQ 0.1 μg/kg were determined, for HT-2 toxin, LOD was 0.25 μg/kg and LOQ 0.8 μg/kg. The precision and accuracy for T-2 and HT-2 toxins were verified at three concentration levels; 25 μg/kg, 100 μg/kg and 500 μg/kg; triplicate. Recoveries for T-2 toxin varied in the range 96.7–106.9% with RSDs ≤ 7.6% (n = 3) and for HT-2 in the range 71.6–78.8% with RSDs ≤ 9.9% RSD (n = 3).
Validity of the method was also verified via interlaboratory comparative trials organized by Central Institute for Supervising and Testing in Agriculture (ÚKZÚZ Brno, the Czech Republic). Six laboratories successfully participated in the testing for T-2 toxin and HT-2 toxin in ground milk thistle seeds with standard z-score −1.53–1.40 and −1.46–1.02, respectively. Obtained values varied in the range 39.3–77.4 μg/kg and 34.5–62.2 μg/kg, respectively; the average values were 59.1 μg/kg and 50.8 μg/kg, respectively.

2.2. Determination of T-2 and HT-2 Toxins in Milk Thistle by UPLC-MS/MS

The content of T-2 toxin in the samples from 2020 was in the range of 122.7–290.2 µg/kg and HT-2 toxin 157.0–319.0 µg/kg. The average sum of T-2 and HT-2 toxins was 389.4 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8–69.9 µg/kg and HT-2 toxin was 24.2–75.4 µg/kg. The average sum of T-2 and HT-2 was 97.5 µg/kg. The obtained results indicate that the content of T-2 and HT-2 toxins was statistically significantly higher in samples from the harvest year 2020, compared to the harvest year 2021. The results of this study are shown in Table 1 and Table 2. The T-2 toxin content was statistically significantly highest in the 2020 samples in Variant 12.5 B: 284.8 µg/kg. In contrast, the statistically significantly lowest content was found in the samples of Variant 25 C: 124.3 µg/kg. However, these samples were not statistically significantly different from the samples of Variant 25 A: 125.4 µg/kg. In 2020, the statistically significantly highest HT-2 toxin content was found in the samples of Variant 37 D: 313.9 µg/kg. The statistically significantly lowest HT-2 toxin content in 2020 was found in the samples of Variant 25 C: 157.8 µg/kg.
In the year 2021, the statistically significantly highest T-2 toxin content was found in the samples of Variant 12.5 B: 69.5 µg/kg. These results were not statistically significantly different from the samples of Variant 37 C: 65.8 µg/kg. In contrast, the statistically significantly lowest T-2 toxin content was found in the samples of Variant 25 A and D: 29.2 and 33.3 µg/kg, respectively, which were not statistically significantly different from the samples of Variant 12.5 A: 33.8 µg/kg. The statistically significantly highest HT-2 toxin content was detected in the samples of Variant 25 D: 87.7 µg/kg. On the other hand, the statistically significantly lowest HT-2 content was also detected in the samples of Variant 25 A: 25.0 µg/kg. However, these results were not statistically significantly different from the samples of Variant 37 A: 28.1 µg/kg. Table 2 shows that the majority of the 2020 samples were found to have higher levels of HT-2 toxin, with the exception of the Variant 12.5 B sample, which had higher levels of T-2 toxin. Compared to 2020, in the year 2021, the T-2 toxin content was higher in more samples, especially in Variant 25 A, B and C (29.2, 58.2 and 56.9 µg/kg, respectively). Sample D of the same Variant 2 showed several times higher HT-2 toxin content (87.7 µg/kg) compared to T-2 toxin content (33.3 µg/kg). At the same time, the HT-2 toxin content was statistically significantly the highest among all the samples from the year 2020. Furthermore, the T-2 toxin content was higher in the samples of milk thistle Variant 37 A, B, C and D (44.9, 39.0, 65.8 and 47.9 µg/kg, respectively).
The analysis of variance showed that the effect of year on T-2 and HT-2 mycotoxin content in milk thistle samples in this study was statistically very highly significant, as well as the effect of individual variants of the randomized trial with different row widths (n = 4 for each Variant). From subsequent testing, it can be concluded that the repetition that was located in the position at the plot edge could have shown higher mycotoxin content.

3. Discussion

Milk thistle has been examined for mycotoxin contamination and reports of research carried out showed some cases of significant levels of contamination, in particular by aflatoxins, deoxynivalenol, T-2 and HT-2 toxins, exceeding the allowed maximum limits. The results were primarily found in dietary supplements [9,12,14,25,26,27,48].
Santos et al. [25] presented a study of medicinal and aromatic plants obtained from Spain, monitored for possible mycotoxin contamination, including two milk thistle samples, both contaminated by T-2 toxin (17.5 µg/kg and 35.6 µg/kg). HT-2 toxin has not been analysed. Arroyo-Manzanares et al. [26] developed a multiclass QuEChERS method for the analysis of mycotoxins in milk thistle. Different samples of commercial milk thistle purchased in local markets from Spanish Granada were analysed to show the applicability of the method; two samples gave positive results for T-2 toxin (363.0 µg/kg and 453.9 µg/kg) and HT-2 toxin (826.9 µg/kg and 943.7 µg/kg). Veprikova et al. [14] developed a multiclass QuEChERS method for 57 various mycotoxins for herb-based supplements. In total, 32 milk thistle-based supplements from Czech and US retail markets for treatment of liver diseases were examined. The level of maximal concentration of T-2 toxin was 1870 µg/kg and HT-2 toxin 1530 µg/kg. Highest total mycotoxin content found in milk thistle-based supplements reached 37 mg/kg. Fenclova et al. [27] studied the composition, chemical and biological safety of 26 milk thistle-based dietary supplements purchased from United States and Czech Republic markets in 2016–2017. The most frequent mycotoxins found in the milk thistle-based preparations were the Fusarium mycotoxins. Type A trichothecenes, T-2 and HT-2, toxins occurred in 92% and 96% of the samples, respectively. Pickova et al. [9] monitored the content of mycotoxins in milk thistle-based supplements; T-2 toxin was detected in 52 of 67 food supplement samples. The highest amounts of T-2 toxin were found in capsules with dried powder (5958 µg/kg) and seeds (453.9 µg/kg). HT-2 toxin was present in 48 of 65 dietary supplement samples. The maximum levels of HT-2 toxin were found in capsules with dried powder (2985 µg/kg) and seeds (943.7 µg/kg).
As previously reported, milk thistle achenes tend to contain not only bioactive substances favourable for human and animal health, such as the silymarin complex, but also naturally occurring contaminants: mycotoxins produced as secondary metabolites by microscopic filamentous micromycetes [27]. The occurrence of mycotoxins in food and feed is still a problem of utmost importance [49]. Higher levels of mycotoxins in raw materials for the pharmaceutical industry or the production of dietary supplements may be influenced by several factors. In the case of milk thistle, indeterminate growth is problematic in terms of cultivation technology, as it causes uneven flowering and maturation of seeds on the plant [50]. It is rather common for milk thistle that mature seeds fall out from the anthodia prematurely or are eaten by birds. Proper timing of the harvest is therefore crucial, and it is quite difficult to determine because of the uneven ripening of the achenes [50,51]. Soil and climate conditions during cultivation are another important factor in the development of fusariosis or other spread of other mycotoxin producers [51,52]. Inappropriate post-harvest treatment can be another undesirable factor, especially insufficient drying; after harvesting the achenes must be cleaned and dried at 50 °C to moisture content ≤ 8 % [51]. The presence of mycotoxins could be controlled by cultivation technologies; however, the use of pesticides is not allowed for this commodity as it is not in a good accordance with a current trend of global pesticide use reduction. In this study, only herbicide treatments were used early in the development of milk thistle plants. Another very important task is increase the awareness of milk thistle growers to mycotoxin producers to encourage them to pay more attention to the quality of raw materials. The occurrence of mycotoxin pathogens can be significantly influenced by the configuration of cultivation technologies [53].
In the European Union, maximum limits have not been set yet for the mycotoxin content in milk thistle for pharmaceutical industry or food supplements. However, the concentrations determined in this work indicate that the values often exceed maximum limits set for other commodities, such as cereals. According to the standard EC 165/2013 the legally recommended maximum levels for the sum of T-2 and HT-2 toxins in cereals intended for human consumption is 200 μg/kg [54]. The sum of T-2 and HT-2 toxins in Sample 12.5 B from 2020 (avg. 562.4 μg/kg) exceeded the limit 2.8-fold. This comparison of mycotoxin concentrations determined in milk thistle samples with maximum limits given for cereals is of course purely theoretical, as the maximum limits take into account not only toxicity but also the amount of food that is normally consumed [14]. The best way to express the risk associated with mycotoxins in milk thistle is to calculate the daily exposure based on the recommended dosage and compare such calculated value with the tolerable daily intake (TDI) given by the regulations. The daily intake of mycotoxins calculated on the basis of the recommended food consumption should be considered to assess the potential health risk associated with the occurrence of mycotoxins. Highest daily exposure is due to the consumption of contaminated milk thistle achenes [14,27]. The most common use of milk thistle is direct oral consumption in the form of ground achenes, standardized tablets, capsules or extracts containing 200–400 mg of silymarin complex. Consumption of milk thistle in the form of tea is highly discouraged because silymarin is only slightly soluble in water [9,55]. Recommended daily dose of freshly ground seeds is up to 15 g (2 tablespoons) per day [55] and the TDI has been set at 0.02 μg/kg body weight per day for the sum of T-2 and HT-2 toxins [47]. Thus, the uptake of the T-2 and HT-2 toxins can reach up to 600% TDI for a person that weights 70 kg (in Sample 12.5 B from 2020, sum of T-2 and HT-2 toxins was 562.4 μg/kg). For the least contaminated Sample 25 B from 2021, containing only 54.2 µg/kg, the uptake reached 80% of TDI. Consumption of Samples 12.5 C and 37 D from the 2021 harvest would cause 130% of TDI. Consumption of such contaminated seeds is highly undesirable as T-2 toxin and its non-acetylated form HT-2 toxin show immunotoxic or hepatotoxic effects on the organism. The toxic impact may be even more severe if dietary supplements of multiple plant constituents with high concentrations of several mycotoxins are consumed, where a so-called mycotoxin “cocktail” is formed [14].
Based on the recommended dosage, none of the monitored samples from the 2020 harvest would have met the limit; however, this could be caused by atypical rainfall [56]. Published data shows that certain weather conditions favour the growth of Fusarium moulds and that contamination of milk thistle with Fusarium mycotoxins is possible, especially during rainy and warmer periods [53,57,58,59]. Figure 2 shows that in 2020, the rainfall was intense mainly in February, June and August (96.2 mm, 136.5 mm and 164.5 mm). In August, before harvest, average rainfall was relatively high, which was unfavourable for harvesting techniques. Figure 3 shows that in in 2021, average rainfall was more similar to the long-term average compared to 2020, except for September and October when rainfall decreased significantly between August (90.3 mm) and September (17.5 mm) in and October (10.7 mm) it was 35 mm less than the long-term average. The average temperature in 2020 was higher than the long-term mean value, with a slight exception in May. The results show that from winter to spring (January–April) the temperatures were higher than the long-term average. In August and October, temperatures were about 2 °C higher. In 2021, the measured temperatures followed the long-term average curve, except for June and July, when temperatures were also higher than in 2020 (avg. 18.7 °C and 19.6 °C).

4. Materials and Methods

4.1. Standards and Chemicals

Solvent purity LC-MS grade as methanol (MeOH), acetonitrile (ACN), 2-propanol (IPA), acetone (ACE), dimethylsulfoxide (DMSO) and standards of mycotoxins T-2 toxin and HT-2 toxin were purchased from Sigma-Aldrich (Prague, Czech Republic). The stock solution standards of mycotoxins (100 µg/mL in acetonitrile) were prepared in 10% methanol/water (v/v) at a concentration of 1 µg/mL and stored at 4 °C in the refrigerator before use. Sodium chloride was purchased from Lachner (Neratovice, Czech Republic). Additives for LC-MS (purity LC-MS) as ammonium acetate, formic acid, acetic acid and citric acid (purity ≥ 99.5%) were purchased from Sigma-Aldrich (Prague, Czech Republic). Check-Sample-Survey, milk thistle powder obtained from Central Institute for Supervising and Testing in Agriculture (ÚKZÚZ Brno, Czech Republic). Ultrapure water was produced by Aqua Osmotic 06 (Tišnov, Czech Republic). Immunoaffinity columns R-Biopharm EASI-EXTRACT® T-2 & HT-2 were obtained from Jemo Trading (Bratislava, Slovak Republic).

4.2. Samples Collection

Randomized milk thistle cultivation experiment was established in experimental station Agritec Plant Research Plc. in Šumperk in the Czech Republic in 2020 and 2021. Individual cultivation variants were carried out with different row widths—12.5 cm, 25 cm and 37 cm with n = 4 (A, B, C and D). The amount of sown seeds was 8 kg/ha. Sowing dates were 25 April 2020 and 30 April 2021. The crops were treated two times with registered herbicides, active ingredients were ethofumesate and in one case also quizalofop-p-ethyl. First treatments were carried out on 22 May 2020 and 25 May 2021, respectively, in the phase of 2 true leaves, second treatments were done on 1 June 2020 and 5 June 2021, respectively, in rosette phase. Harvest times were 9 September 2020 and 25 August 2021. The yields of achenes were 0.703 t/ha and 1.194 t/ha in 2020 and 2021, respectively. Average rainfall in 2020 was 76.53 mm and in 2021 it was 52.11 mm. Average temperatures were 10.42 °C and 9.24 °C, respectively. Meteorological data was obtained from meteorological station located in Agritec Plant Research Plc., Šumperk, Czech Republic (49°58′26.8″ N 16°58′03.2″ E).
A total of 24 milk thistle [Silybum marianum (L.) Gaertn.] seed samples of the variety Mirel with a weight of 150 g were taken from each sample. Mirel is a legally protected variety, the rights holder is Moravol Ltd. This variety has a high content of good quality oil with a specific fatty acid composition. It is used mostly for the isolation of silymarin complex, but the oil is also pressed from the seeds as a by-product.
The experiment was harvested with a small parcel harvester Sampo Rosenlew SR2010. Due to unfavourable weather conditions a large amount of moisture-increasing impurities was present in the harvested product at the time of harvest. Because of that, pre-cleaning was carried out immediately after the harvest and the harvested material was subsequently transferred to cold air drying.
The samples were cleaned and stored in paper bags in the dark under laboratory conditions: 22 °C and 42% relative humidity.

4.3. Sample Preparation and Clean-Up by Immunoaffinity Columns

Twenty-five grams of milk thistle, finely ground by a grinder AR 1105 (Moulinex, France), were weighed into a Duran Youtility® bottle (Sigma-Aldrich, Prague, Czech Republic) with five grams of sodium chloride, added 125 mL 90% methanol/water (v/v) and shook for 50 min on orbital shaker IKA KS 260 (VWR, Stříbrná Skalice, Czech Republic). After centrifugation at 4800 rpm for 10 min, 20 mL of extract was diluted with 80 mL 2% sodium chloride and left on the bench for 5 min with occasional gently shaking the solution to allow precipitation to occur. Supernatant was filtered through Whatman No. 4 filter paper. The 25 mL aliquot of diluted filtrate was passed through the immunoaffinity column EASI-EXTRACT® T-2 & HT-2, which was inserted into the Supelco SPE Vacuum Manifold Visiprep™ (Sigma-Aldrich, Prague, Czech Republic) at a gravitation flow rate. Slowly and steady flow rate is essential for the capture of the toxins by the antibody. Column was washed by passing 20 mL of water through at a flow rate approximately 5 mL/min, then passed air though the column to remove residual liquid. The toxins were eluted from the column at a flow rate of 1 drop per second using 2 mL 100% methanol and collected in a heart shaped flask. This eluate was evaporated to dry under vacuum in rotary evaporator IKA RV 10 (VWR, Stříbrná Skalice, Czech Republic). The residues were dissolved in 1 mL of 10% methanol/water (v/v) for UPLC-MS/MS analysis according to validated method (n = 2).

4.4. UPLC-MS/MS Mycotoxin Analysis

The Waters Acquity™ H-Class Plus UPLC® system coupled to the Xevo TQ-S Micro mass spectrometer (Waters, Prague, Czech Republic) with electro-spray ionization (ESI) was used for the identification and quantification of mycotoxins. Data acquisition and processing were performed with MassLynx™ version 4.2 software (Waters, Prague, Czech Republic). Chromatographic separation was performed on a column Waters Acquity™ UPLC® BEH C18 (100 × 2.1 mm, 1.7 µm) and the column temperature was maintained at 40 °C using gradient elution. The mobile phase consisted of eluent A (1 mM ammonium acetate + 0.5% acetic acid + 0.1% formic acid in water) and eluent B (0.5% acetic acid + 0.1% formic acid in methanol). The gradient program was applied at a flow rate of 0.4 mL/min under the following conditions: 0 min 90% A; 3.0 min 90% A; 10.0 min 30% A; 10.1 min 10% A; 12.0 min 10% A; 13.1 min 90% A; 15.0 min 90% A. The injection volume was 5 µL and injection mode was a flow-through needle. The needle wash consisted of 7.44 mM citric acid in water:MeOH:ACN:IPA:ACE:DMSO (37:9:19:19:9:7), respectively. Seal wash and purge wash was 10% IPA. The mass spectrometer was operated at ESI and parameters were set up as follows: capillary voltage 30 kV, source temperature and desolvation temperature were 120 °C and 450 °C, respectively. Collision gas was argon and nitrogen applied as spray gas: cone gas flow 100 L/Hr, desolvation gas flow 800 L/Hr. For selectivity, the mass spectrometer was operated in MRM mode and two transitions per mycotoxin were monitored.

4.5. Development, Optimization and Validation of Method

This section describes development, optimalization and validation the data generated by an Acquity™ H-Class plus with a Xevo TQ-S Micro mass spectrometer system for the analysis of T-2 toxin and HT-2 toxin using electrospray ionization. A UPLC method was created to analyse monitored compounds in a 15 min gradient and two MRM transitions (multiple reaction monitoring) were optimized for each analyte using positive or negative electrospray ionization (ESI). The optimized MRM parameters are shown in Table 3.
Linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy of the method were evaluated. Linearity was evaluated using standard solution in seven concentration levels. The LODs were determined as the lowest detectable mycotoxin concentration at which a signal-to-noise ratio (S/N) = 3. The LOQs were determined as the minimum amount of analyte for quantification with a S/N = 10. The accuracy and precision for mycotoxin compounds were determined at three concentration levels for three times. The calibration curves were linear with a good linearity where the coefficient of determination (R2 > 0.999) for each analyte.

4.6. Statistical Analysis

The obtained results were evaluated by one-way analysis of variance (ANOVA) followed by testing at a 95% (p < 0.05) level of significance using the Fisher LSD test. The data were processed using the STATISTICA.CZ version 12.0 software (StatSoft, Prague, Czech Republic). Results are expressed as a mean value ± standard deviation (SD).

5. Conclusions

The safety and quality of food and food raw materials is currently a highly discussed topic. Especially in crops that should bring health benefits, the content of undesirable compounds, harmful to human health, should be controlled. Milk thistle is an important medicinal plant, used mostly in the pharmaceutical industry, and its bioactive compounds help the detoxication of an organism. Thus, especially in these conditions, the occurrence of toxins in such a commodity is extremely alarming. The content of unwanted toxic compounds can be regulated by good agricultural practice and post-harvest treatment. However, the current situation is further complicated by the fact that there is no pesticide preparation effective against these mycotoxin producers legally registered at the moment. The toxicological impact of many mycotoxins that can be found in plants is still unknown, especially when they are present in mixtures, known as “mycotoxin cocktails”; thus, it is important to continue their further monitoring.
Due to the amount of T-2 and HT-2 toxins in some milk thistle samples exceeding 600% of the TDI, it is clear that high health risks can be expected, especially for people with weakened immune systems or liver diseases. Although it is not possible to completely eliminate mycotoxins from milk thistle or other plants, it would be advisable to set their maximum limits. For oilseeds, which include milk thistle, no limits have yet been incorporated into legislation, but standard limit values are already legislatively given for cereals, for example.

Author Contributions

Conceptualization, R.B., H.P., M.P. and S.B.; methodology R.B., H.P., M.P. and S.B.; validation M.P.; formal analysis and final interpretation, M.P. and H.P.; investigation, R.B., M.P. and H.P.; resources, R.B. and H.P.; data curation, R.B. and M.P.; writing—original draft preparation, R.B. and H.P.; writing—review and editing, R.B., H.P. and M.P.; supervision, H.P. and S.B.; setting up an experiment, H.P. and M.B.; project administration, H.P. and S.B.; funding acquisition, H.P., M.B. and S.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Agriculture of the Czech Republic, within Institutional Support MZe-RO1918 and NAZV MZe QK1910302.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request, please contact the contributing authors.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Habán, M.; Habánová, M.; Otepka, P.; Kobida, Ľ. Milk thistle (Silybum marianum L. Gaertn.) cultivated in polyfunctional crop rotation and its evaluation. Res. J. Agric. Sci. 2010, 42, 111–117. [Google Scholar]
  2. Habán, M.; Luščáková, D.; Macak, M.; Ražná, K. The Impact of Multifunctional Crop Rotation on the Yield of Milk Thistle Fruits in the Years 2012–2015. J. Cent. Eur. Agric. 2016, 17, 1096–1103. [Google Scholar] [CrossRef]
  3. European Pharmacopoeia. European Pharmacopoeia, 6th ed.; Council of Europe: Strasbourg, France, 2008; Volume 2, pp. 2425–2427. [Google Scholar]
  4. Morazzoni, P.; Bombardelli, E. Silybum marianum (Carduus marianus). Fitoterapia 1995, 66, 6–42. [Google Scholar]
  5. Saller, R.; Meier, R.; Brignoli, R. The use of silymarin in the treatment of liver diseases. Drugs 2001, 61, 2035–2063. [Google Scholar] [CrossRef]
  6. Habán, M.; Otepka, P.; Habánová, M. Production and quality of milk thistle (Silybum marianum L. Gaertn.) cultivated in cultural conditions of warm agri-climatic macroregion. Hort. Sci. 2009, 36, 69–74. [Google Scholar] [CrossRef] [Green Version]
  7. Škottová, N.; Krečman, V. Silymarin as a potential hypocholesterolaemic drug. Physiol. Res. 1998, 47, 1–7. [Google Scholar]
  8. Wellington, K.; Jarvis, B. Silymarin: A review of its clinical properties in the management of liver disorders. BioDrugs 2001, 15, 465–489. [Google Scholar] [CrossRef]
  9. Pickova, D.; Ostry, V.; Toman, J.; Malir, F. Presence of Mycotoxins in Milk Thistle (Silybum marianum) Food Supplements: A Review. Toxins 2020, 12, 782. [Google Scholar] [CrossRef]
  10. Singh, R.P.; Deep, G.; Chittezhath, M.; Kaur, M.; Dwyer-Nield, L.D.; Malkinson, A.M.; Agarwal, R. Effect of silibinin on the growth and progression of primary lung tumors in mice. J. Natl. Cancer Inst. 2006, 98, 846–855. [Google Scholar] [CrossRef] [Green Version]
  11. Valková, V.; Ďúranová, H.; Bilčíková, J.; Habán, M. Milk thistle (Silybum marianum): A valuable medicinal plant with several therapeutic purposes. J. Microbiol. Biotechnol. Food Sci. 2021, 2021, 836–843. [Google Scholar] [CrossRef]
  12. Tournas, V.H.; Sapp, C.; Trucksess, M.W. Occurrence of aflatoxins in milk thistle herbal supplements. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2012, 29, 994–999. [Google Scholar] [CrossRef] [PubMed]
  13. Kosalec, I.; Cvek, J.; Tomic, S. Contaminants of medicinal herbs and herbal products. Arh. Hig. Rada Toksikol. 2009, 60, 485–501. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Veprikova, Z.; Zachariasova, M.; Dzuman, Z.; Zachariasova, A.; Fenclova, M.; Slavikova, P.; Vaclavikova, M.; Mastovska, K.; Hengst, D.; Hajslova, J. Mycotoxins in plant-based dietary supplements: Hidden health risk for consumers. J. Agri. Food Chem. 2015, 63, 6633–6643. [Google Scholar] [CrossRef] [PubMed]
  15. Tassaneeyakul, W.; Razzazi-Fazeli, E.; Porasuphatana, S.; Bohm, J. Contamination of aflatoxins in herbal medicinal products in Thailand. Mycopathologia 2004, 158, 239–244. [Google Scholar] [CrossRef]
  16. Cao, H.; Huang, H.; Xu, W.; Chen, D.; Yu, J.; Li, J.; Li, L. Fecal metabolome profiling of liver cirrhosis and hepatocellular carcinoma patients by ultra performance liquid chromatography-mass spectrometry. Anal. Chim. Acta 2011, 691, 68–75. [Google Scholar] [CrossRef]
  17. Posadzki, P.; Watson, L.; Ernst, E. Contamination and adulteration of herbal medicinal products (HMPs): An overview of systematic reviews. Eur. J. Clin. Pharm. 2013, 69, 295–307. [Google Scholar] [CrossRef]
  18. Pitt, J.I.; Hocking, A.D. Fungi and Food Spoilage, 3rd ed.; Springer: Boston, MA, USA, 2009; pp. 89–122. [Google Scholar]
  19. Běláková, S.; Benešová, K.; Čáslavský, J.; Svoboda, Z.; Mikulíková, R. The occurrence of the selected Fusarium mycotoxins in Czech malting barley. Food Contr. 2014, 37, 93–98. [Google Scholar] [CrossRef]
  20. Zhang, J.; Zhang, H.; Liu, S.; Wu, W.; Zhang, H. Comparison of anorectic potencies of type a trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and neosolaniol. Toxins 2018, 10, 179. [Google Scholar] [CrossRef] [Green Version]
  21. Beccari, G.; Caproni, L.; Tini, F.; Uhlig, S.; Covarelli, L. Presence of Fusarium species and other toxigenic fungi in malting barley and multi-mycotoxin analysis by liquid chromatography–high resolution mass spectrometry. J. Agric. Food Chem. 2016, 64, 4390–4399. [Google Scholar] [CrossRef]
  22. Edwards, S.G.; Imathiu, S.M.; Ray, R.V.; Back, M.; Hare, M.C. Molecular studies to identify the Fusarium species responsible for HT-2 and T-2 mycotoxins in UK oats. Int. J. Food Microbiol. 2012, 156, 168–175. [Google Scholar] [CrossRef]
  23. Pernica, M.; Kyralová, B.; Svoboda, Z.; Boško, R.; Brožková, I.; Česlová, L.; Benešová, K.; Červenka, L.; Běláková, S. Levels of T-2 toxin and its metabolites, and the occurrence of Fusarium fungi in spring barley in the Czech Republic. Food Microbiol. 2022, 102, 103875. [Google Scholar] [CrossRef] [PubMed]
  24. Malachova, A.; Cerkal, R.; Ehrenbergerova, J.; Dzuman, Z.; Vaculova, K.; Hajslova, J. Fusarium mycotoxins in various barley cultivars and their transfer into malt. J. Sci. Food Agric. 2010, 90, 2495–2505. [Google Scholar] [CrossRef] [PubMed]
  25. Santos, L.; Marín, S.; Sanchis, V.; Ramos, A.J. Screening of mycotoxin multicontamination in medicinal and aromatic herbs sampled in Spain. J. Sci. Food Agric. 2009, 89, 1802–1807. [Google Scholar] [CrossRef]
  26. Fenclova, M.; Novakova, A.; Viktorova, J.; Jonatova, P.; Dzuman, Z.; Ruml, T.; Kren, V.; Hajslova, J.; Vitek, L.; Stranska-Zachariasova, M. Poor chemical and microbiological quality of the commercial milk thistle-based dietary supplements may account for their reported unsatisfactory and non-reproducible clinical outcomes. Sci. Rep. 2019, 9, 1–12. [Google Scholar]
  27. Arroyo-Manzanares, N.; García-Campaña, A.M.; Gámiz-Gracia, L. Multiclass mycotoxin analysis in Silybum marianum by ultra-high performance liquid chromatography–tandem mass spectrometry using a procedure based on QuEChERS and dispersive liquid–liquid microextraction. J. Chromatogr. A 2013, 1282, 11–19. [Google Scholar] [CrossRef]
  28. Pernica, M.; Piacentini, K.C.; Benešová, K.; Čáslavský, J.; Běláková, S. Analytical techniques for determination of mycotoxins in barley, malt and beer: A review. Kvas. Prum. 2019, 65, 46–57. [Google Scholar] [CrossRef]
  29. Li, Y.; Wang, Z.; Beier, R.C.; Shen, J.; Smet, D.D.; De Saeger, S.; Zhang, S. T-2 toxin, a trichothecene mycotoxin: Review of toxicity, metabolism, and analytical methods. J. Agric. Food. Chem. 2011, 59, 3441–3453. [Google Scholar] [CrossRef]
  30. Karron, E.; Runno-Paurson, E.; Lõiveke, H.; Islamov, B.; Kütt, M.L.; Talve, T.; Lauringson, E.; Horak, H.; Edesi, L.; Niinemets, Ü. Application of widely used fungicides does not necessarily affect grain yield, and incidence of Fusarium spp. and mycotoxins DON, HT-2 and T-2 in spring barley in northern climates. Kvas. Prum. 2020, 66, 215–223. [Google Scholar] [CrossRef] [Green Version]
  31. Mateo, J.J.; Mateo, R.; Jimenez, M. Accumulation of type A trichothecenes in maize, wheat and rice by Fusarium sporotrichioides isolates under diverse culture conditions. Int. J. Food. Microbiol. 2002, 72, 115–123. [Google Scholar] [CrossRef]
  32. Jesenska, Z.; Sajbidorova, I. T-2 toxin degradation by micromycetes. J. Hyg. Epidemiol. Microbiol. Immunol. 1991, 35, 41–49. [Google Scholar]
  33. Yang, X.; Liu, P.; Cui, Y.; Xiao, B.; Liu, M.; Song, M.; Huang, W.; Li, Y. Review of the reproductive toxicity of T-2 toxin. J. Agric. Food Chem. 2020, 68, 727–734. [Google Scholar] [CrossRef] [PubMed]
  34. Čonková, E.; Laciaková, A.; Kováč, G.; Seidel, H. Fusarial toxins and their role in animal diseases. Vet. J. 2003, 165, 214–220. [Google Scholar] [CrossRef]
  35. Doi, K.; Ishigami, N.; Sehata, S. T-2 toxin-induced toxicity in pregnant mice and rats. Int. J. Mol. Sci. 2008, 9, 2146–2158. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Rafai, P.; Bata, A.; Vanyi, A.; Papp, Z.; Brydl, E.; Jakab, L.; Tuboly, S.; Tury, E. Effect of various levels of T-2 toxin on the clinical status, performance and metabolism of growing pigs. Vet. Rec. 1995, 136, 485–489. [Google Scholar] [CrossRef]
  37. Dai, C.; Xiao, X.; Sun, F.; Zhang, Y.; Hoyer, D.; Shen, J.; Tang, S.; Velkov, T. T-2 toxin neurotoxicity: Role of oxidative stress and mitochondrial dysfunction. Arch. Toxicol. 2019, 93, 3041–3056. [Google Scholar] [CrossRef]
  38. Shinozuka, J.; Suzuki, M.; Noguchi, N.; Sugimoto, T.; Uetsuka, K.; Nakayama, H.; Doi, K. T-2 toxin-induced apoptosis in hematopoietic tissues of mice. Toxicol. Pathol. 1998, 26, 674–681. [Google Scholar] [CrossRef]
  39. Li, D.; Han, J.; Guo, X.; Qu, C.; Yu, F.; Wu, X. The effects of T-2 toxin on the prevalence and development of Kashin–Beck disease in China: A meta-analysis and systematic review. Toxicol. Res. 2016, 5, 731–751. [Google Scholar] [CrossRef] [Green Version]
  40. Visconti, A. Problems associated with Fusarium mycotoxins in cereals. Bull. Inst. Compr. Agric. Sci. Kinki. Univ. 2001, 9, 39–55. [Google Scholar]
  41. Van der Fels-Klerx, H.J.; Stratakou, I. T-2 toxin and HT-2 toxin in grain and grain-based commodities in Europe: Occurrence, factors affecting occurrence, co-occurrence and toxicological effects. World Mycotoxin J. 2010, 3, 349–367. [Google Scholar] [CrossRef]
  42. Canady, R.A.; Coker, R.D.; Egan, S.; Krska, R.; Olsen, M.; Resnik, S.; Schlatter, J. T-2 and HT-2 toxins. Saf. Eval. Certain Mycotoxins Food. WHO Food Addit. Ser. 2001, 47, 557–597. [Google Scholar]
  43. Wu, Q.; Dohnal, V.; Huang, L.; Kuča, K.; Yuan, Z. Metabolic pathways of trichothecenes. Drug Metab Rev. 2010, 42, 250–267. [Google Scholar] [CrossRef]
  44. Daud, N.; Currie, V.; Duncan, G.; Busman, M.; Gratz, S.W. Intestinal hydrolysis and microbial biotransformation of diacetoxyscirpenol-α-glucoside, HT-2-β-glucoside and N-(1-deoxy-D-fructos-1-yl) fumonisin B1 by human gut microbiota in vitro. Int. J. Food Sci. Nutr. 2020, 71, 540–548. [Google Scholar] [CrossRef] [PubMed]
  45. EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed. EFSA J. 2011, 9, 2481. [Google Scholar] [CrossRef]
  46. Wu, W.; Zhou, H.R.; Pan, X.; Pestka, J.J. Comparison of anorectic potencies of the trichothecenes T-2 toxin, HT-2 toxin and satratoxin G to the ipecac alkaloid emetine. Toxicol. Rep. 2015, 2, 238–251. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. EFSA Panel on Contaminants in the Food Chain (CONTAM). Appropriateness to set a group health based guidance value for T-2 and HT-2 toxin and its modified forms. EFSA J. 2017, 15, e04655. [Google Scholar]
  48. Ałtyn, I.; Twarużek, M. Mycotoxin Contamination Concerns of Herbs and Medicinal Plants. Toxins 2020, 12, 182. [Google Scholar] [CrossRef] [Green Version]
  49. Steiner, D.; Malachová, A.; Sulyok, M.; Krska, R. Challenges and future directions in LC-MS-based multiclass method development for the quantification of food contaminants. Anal. Bioanal. Chem. 2021, 413, 25–34. [Google Scholar] [CrossRef]
  50. Carrier, D.J.; Crowe, T.; Sokhansanj, S.; Wahab, J.; Barl, B. Milk thistle, Silybum marianum (L.) Gaertn., flower head development and associated marker compound profile. J. Herbs. Spices Med. Plants 2003, 10, 65–74. [Google Scholar] [CrossRef]
  51. Andrzejewska, J.; Sadowska, K.; Mielcarek, S. Effect of sowing date and rate on the yield and flavonolignan content of the fruits of milk thistle (Silybum marianum L. Gaertn.) grown on light soil in a moderate climate. Ind. Crops Prod. 2011, 33, 462–468. [Google Scholar] [CrossRef]
  52. Champeil, A.; Doré, T.; Fourbet, J.F. Fusarium head blight: Epidemiological origin of the effects of cultural practices on head blight attacks and the production of mycotoxins by Fusarium in wheat grains. Plant Sci. 2004, 166, 1389–1415. [Google Scholar] [CrossRef]
  53. Kiš, M.; Vulić, A.; Kudumija, N.; Šarkanj, B.; Jaki Tkalec, V.; Aladić, K.; Škrivanko, M.; Furmeg, S.; Pleadin, J. A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather. Toxins 2021, 13, 39. [Google Scholar] [CrossRef] [PubMed]
  54. European Union. 2013/165/EU Commission Recommendation of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal product. Off. J. Eur. Union 2013, L91, 12–15. [Google Scholar]
  55. Bhattacharya, S. Milk thistle seeds in health. In Nuts and Seeds in Health and Disease Prevention; Academic Press: London, UK, 2020; pp. 429–438. [Google Scholar]
  56. Imathiu, S.M.; Edwards, S.G.; Ray, R.V.; Back, M.A. Fusarium langsethiae—A HT-2 and T-2 toxins producer that needs more attention. J. Phytopathol. 2013, 161, 1–10. [Google Scholar] [CrossRef]
  57. Pleadin, J.; Vahčić, N.; Perši, N.; Ševelj, D.; Markov, K.; Frece, J. Fusarium mycotoxins’ occurrence in cereals harvested from Croatian fields. Food Control 2013, 32, 49–54. [Google Scholar] [CrossRef]
  58. Kochiieru, Y.; Mankevičienė, A.; Cesevičienė, J.; Semaškienė, R.; Dabkevičius, Z.; Janavičienė, S. The influence of harvesting time and meteorological conditions on the occurrence of Fusarium species and mycotoxin contamination of spring cereals. J. Sci. Food Agric. 2020, 100, 2999–3006. [Google Scholar] [CrossRef] [PubMed]
  59. Kochiieru, Y.; Mankevičienė, A.; Cesevičienė, J.; Semaškienė, R.; Ramanauskienė, J.; Gorash, A.; Janavičienė, S.; Venslovas, E. The Impact of Harvesting Time on Fusarium Mycotoxins in Spring Wheat Grain and Their Interaction with Grain Quality. Agronomy 2021, 11, 642. [Google Scholar] [CrossRef]
Toxins 14 00258 g001 550
Figure 1. Chemical structure of trichothecenes T-2 and HT-2 toxins.
Figure 1. Chemical structure of trichothecenes T-2 and HT-2 toxins.
Toxins 14 00258 g001
Toxins 14 00258 g002 550
Figure 2. Average precipitation in Šumperk (Czech Republic) during vegetation and harvest period.
Figure 2. Average precipitation in Šumperk (Czech Republic) during vegetation and harvest period.
Toxins 14 00258 g002
Toxins 14 00258 g003 550
Figure 3. Average temperature in Šumperk (Czech Republic) during vegetation and harvest period.
Figure 3. Average temperature in Šumperk (Czech Republic) during vegetation and harvest period.
Toxins 14 00258 g003
Table
Table 1. ANOVA for the average T-2 and HT-2 toxin content in milk thistle samples from 2020 and 2021.
Table 1. ANOVA for the average T-2 and HT-2 toxin content in milk thistle samples from 2020 and 2021.
Source of VariabilityDFT-2 Toxin [µg/kg] MSHT-2 Toxin [µg/kg] MS
Year1194,669.8 ***317,040.4 ***
Variant112938.6 ***2303.2 ***
Year*variant111972.0 ***2540.0 ***
Error245.818.3
Legend: DF = Degree of Freedom, MS = Mean Square; * p ≤ 0.05; *** p ≤ 0.001.
Table
Table 2. The average T-2 and HT-2 toxins content in samples from 2020 and 2021.
Table 2. The average T-2 and HT-2 toxins content in samples from 2020 and 2021.
YearVariantT-2 Toxin [µg/kg]HT-2 Toxin [µg/kg]
20201. (12.5 cm)A180.3 ± 2.9j238.9 ± 3.4o
B284.8 ± 7.7n277.6 ± 8.4p
C136.5 ± 4.1h172.9 ± 1.2k
D200.2 ± 1.5l204.8 ± 6.5n
2. (25 cm)A125.4 ± 1.3g187.8 ± 5.8lm
B136.5 ± 0.2h188.9 ± 4.6lm
C124.3 ± 2.2g157.8 ± 1.2j
D160.2 ± 5.3i186.6 ± 6.0l
3. (37 cm)A176.1 ± 0.3j239.2 ± 9.8o
B187.1 ± 0.7k201.4 ± 5.1n
C158.9 ± 3.2i196.6 ± 1.7mn
D236.1 ± 1.8m313.9 ± 7.3q
20211. (12.5 cm)A33.8 ± 0.0a38.4 ± 1.1bc
B69.5 ± 0.6f73.2 ± 3.1h
C42.0 ± 0.2bc48.7 ± 1.3de
D57.4 ± 0.5e62.9 ± 1.1fg
2. (25 cm)A29.2 ± 0.4a25.0 ± 1.2a
B58.2 ± 0.4e54.7 ± 2.6ef
C56.9 ± 0.2e53.7 ± 1.6de
D33.3 ± 0.3a87.7 ± 2.5i
3. (37 cm)A44.9 ± 1.5cd28.1 ± 1.8a
B39.0 ± 0.3b33.5 ± 1.5ab
C65.8 ± 0.7f64.7 ± 2.6gh
D47.9 ± 0.2d45.3 ± 0.4cd
Legend: The average values marked with different letters in the rows differ statistically significantly at p = 0.05.
Table
Table 3. Optimized MRM parameters for monitored mycotoxins.
Table 3. Optimized MRM parameters for monitored mycotoxins.
MycotoxinRetention Time
[min]		Precursor Ion
[m/z]		Product Ion
[m/z]		Collision Energy
[V]
T-2 toxin10.66[M + NH4]+185.00 *22
484.25215.15 **22
HT-2 toxin10.03[M + NH4]+215.13 *12
442.17263.15 **12
* quantification ion; ** qualifier ion.
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Boško, R.; Pernica, M.; Běláková, S.; Bjelková, M.; Pluháčková, H. Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS. Toxins 2022, 14, 258. https://doi.org/10.3390/toxins14040258

AMA Style

Boško R, Pernica M, Běláková S, Bjelková M, Pluháčková H. Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS. Toxins. 2022; 14(4):258. https://doi.org/10.3390/toxins14040258

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Boško, Rastislav, Marek Pernica, Sylvie Běláková, Marie Bjelková, and Helena Pluháčková. 2022. "Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS" Toxins 14, no. 4: 258. https://doi.org/10.3390/toxins14040258

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