**2.4. Validation of the analytical methods used**

cereal samples were combined of three, five or ten incremental samples, depending on the lot

44 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

The samples were stored in a cool and dry place and transported to the laboratory within 48 h. The prepared test portions (500 g per sample) were ground into a fine powder having a particle size of 1.0 mm using an analytical mill (Cylotec 1093, Tecator, Sweden), and then

T-2 toxin (Art. No. 34071, 100 μg/mL in acetonitrile) and HT-2 toxin (Art. No. 34136, 100 μg/ mL in acetonitrile) standards were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). A RIDASCREEN® T-2/HT-2 toxin kit (Art. No. R3805) was provided by R-Biopharm (Darmstadt, Germany). PuriTox Total Myco-MS solid phase clean-up columns (Art. No. TC-MT3000) were produced by R-BiopharmRône LTD (Glasgow, Scotland). The Certified Reference oat flour Material (CRM) (Art. No. TET039RM) having the reference values of 85.3 ± 13.7 μg/kg for T-2 toxin and 86.9 ± 11.9 μg/kg for HT-2 toxin, was purchased from Fapas,

All chemicals used for ELISA and LC-MS/MS analyses were of an analytical grade (acetic acid, Art. No. 33209, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) or a HPLC grade (acetonitrile, Art. No. 34851, and methanol, Art. No. 34885 Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Ultrapure water was supplied by the Merck system Direct-Q 3 UV (New Jersey, USA).

All samples were first analysed using the validated ELISA method. After grinding, to 5 g of a homogenised sample, 25 mL of methanol/distilled water solution (70/30; v/v) were added, except for oat samples, to which 25 mL of the appropriate extraction buffer provided with the ELISA kit were added. The extraction of T-2/HT-2 toxin was performed using a headover-head shaker for 10 min (100 rpm, room temperature) followed by an additional 10-min centrifugation (4000 rpm, room temperature). When it comes to oat samples, the obtained supernatant was diluted in methanol/distilled water (70/30; v/v) in 1:2 ratio, while the supernatant obtained with all other biological materials under study was diluted in distilled water in 1:2 ratio. The obtained solutions were then transferred into the wells of the ELISA microtitration plate. The ELISA tests were performed using a ChemWell autoanalyser (Awareness Technology Inc. 2910, Palm City, USA), observing thereby the instructions given by the kit provider. Once the stop solution had been injected, the absorbances were determined at 450 nm. In order to calculate the summary T-2/HT-2 concentration in an individual sample, the results provided by the calibration curve were multiplied by the corresponding sample dilution factor. The calculation of the summary toxin concentrations was guided by the aver-

Samples, in which T-2/HT-2 concentrations higher than the ELISA's Limit of Detection were established, were further analysed using the LC-MS/MS method. To that effect, to 2.5 g of a test sample, 10 mL of 80%-acetonitrile were added and vortexed for 30 s; afterwards, the samples

weight, each lot thereby weighing at least 1 kg.

**2.2. Chemicals, standards and reference materials**

age recovery values ascertained by method validation.

stored at 4°C pending analyses.

Fera Science Ltd. (York, UK).

**2.3. Analytical methods**

For the ELISA method, the limit of detection (LOD) and the limit of quantification (LOQ) were calculated from the mean value of 10 control samples of a given cereal (maize, wheat, oat, barley or triticale) plus 3- and 10-fold standard deviation. For each cereal, the recoveries were determined in three replicates per concentration level per day. To that goal, the control samples were supplemented with 50% T-2 and 50% HT-2 toxin standard working solutions (either 500 μg/L or 1000 μg/L).

As for the LC-MS/MS method, the LOD and LOQ values were estimated according to the Guidance document on the estimation of LOD and LOQ for measurements in the field of contaminants in feed and food [23] via paired observations. In brief, 10 pseudo-blank samples of


A less intense ion—used as a qualifier.

**Table 1.** Ions monitored within the frame of LC-MS/MS analyses targeted at T-2 and HT-2 toxin.

different cereals, i.e. maize, oat, wheat, barley and triticale, were chosen based on the results obtained using the ELISA technique (<LOD) and spiked with the T-2 and HT-2 standard at the level of 25 μg/kg. Then, both spiked and pseudo-blank samples were analysed, and the difference in signal abundances and the standard deviation was calculated. The obtained data were used for the calculation of the LOD and the LOQ. Linearity was tested in the range of 5–100 ng/mL, using the standard solution of each mycotoxin. The recovery was determined by virtue of analysing six blank maize samples spiked with T-2 and HT-2 toxin at 50 μg/kg, while the trueness was tested using six replicates, making use of oat flour as the CRM, and later compared with the values assigned for each mycotoxin by the manufacturer.

**2.6. Data analysis**

significance thereby being set at 95% (p = 0.05).

**3.1. Validation of analytical methods used**

coefficient of variation (CV) of 7.8% (**Table 2**).

individual concentrations of mycotoxins under study.

**3.2. The occurrence of T-2/HT-2 toxin in cereals**

**3. Results and discussion**

0.99 for both analytes (**Table 3**).

Concentrations (μg/kg) obtained by the ELISA assay are expressed as mean summary values (T-2/HT-2) ± standard deviation (SD), while in toxin-positive samples, the concentrations are given as individual concentrations of each mycotoxin (T-2 or HT-2) obtained by the LC-MS/ MS. Statistical analysis was performed using the Statistica ver. 10.0 software (StatSoft Inc. 1984-2011, Tulsa, OK, USA) and made use of the analysis of variance (ANOVA), the statistical

The Incidence of T-2 and HT-2 Toxins in Cereals and Methods of their Reduction Practice by the Food Industry

http://dx.doi.org/10.5772/intechopen.71550

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The ELISA assay, used as a quantitative screening method for the determination of summary concentrations of T-2/HT-2 toxins, was first validated and then applied for the analyses of the sampled cereals. Its cross-reactivity declared by the kit manufacturer is approximately 85% for T-2 toxin and 100% for the HT-2 toxin. Validation of the ELISA method resulted in LODs ranging from 20.6 to 30.1 μg/kg and LOQs ranging from 26.7 to 37.4 μg/kg, depending on the type of cereal under consideration. The mean recovery value equalled to 90.1%, with the mean

Validation of the LC-MS/MS method resulted in LODs spanning from 5.5 to 8.3 μg/kg, and LOQs ranging from 18.2 to 27.5 μg/kg for T-2 and HT-2 toxin, respectively. The mean recovery values were 89.6 and 77.0%, with the mean CV of 5.1% and 8.9%, respectively. The analyses of oat flour sample (CRM) used for the determination of trueness, resulted in concentrations of 77.9 μg/kg for T-2 (91.3% of the mean certified value) and 75.6 μg/kg for HT-2 (86.9% of the mean certified value). **Figure 1** presents a typical LC-MS/MS-MRM chromatogram of the analysed CRM. The determination of linearity resulted in correlation coefficients higher than

Given the obtained validation results, both analytical methods were recognised as suitable for analyses of different cereals, the ELISA assay thereby being employed as a screening method used for the determination of summary concentrations of T-2/HT-2 toxins, and the LC-MS/ MS thereby being used as a confirmatory method exploited to the effect of determination of

Data published insofar have revealed that the exposure to T-2 and HT-2 toxins primarily comes as a result of the consumption of cereal grains and cereal by-products, wherein the levels of these toxins found in forages and oilseed meals are generally low. It has been established that T-2 and HT-2 toxins occur together, HT-2 thereby representing approximately two-thirds of the summary T-2/HT-2 concentration. The highest mean T-2/HT-2 concentrations were determined in grains and milled grain products, in particular in oat and oat-based
