**2.1. Sampling and sample preparation**

During the period spanning from May 2015 to April 2017, a total of 285 samples of unprocessed cereals, in specific maize (n = 84), wheat (n = 56), oat (n = 72), barley (n = 44) and triticale (n = 29), were sampled from households situated in the Northern, Central and Eastern part of Croatia. All cereals were grown in the crop season 2015 and 2016 and had not undergone any physical or thermal treatment other than drying, cleaning and sorting prior to sampling. Sampling and sample preparation of unprocessed cereals were performed in full line with the Commission Regulation 401/2006 [22], laying down the methods of sampling to be exercised within the frame of the official control of mycotoxin levels in foodstuffs. The aggregate cereal samples were combined of three, five or ten incremental samples, depending on the lot weight, each lot thereby weighing at least 1 kg.

were put on a head-over-head shaker for 10 min (100 rpm, room temperature). The samples were then centrifuged (10 min, 4000 rpm, room temperature). Two millilitres of the obtained supernatant were acidified with 20 μL of (glacial) acetic acid; after that, 1.4 mL of the obtained solution was passed through the PuriTox Total Myco-MS columns (R-Biopharm, Glasgow, Scotland). Five hundred microliters were then evaporated under a nitrogen stream at 40°C and reconstituted in 250 μL of 1%-acetic acid in 20% acetonitrile. Fifty microliters were injected onto the LC-MS/MS system. The LC-MS/MS system consisted of a degasser, a binary pump, an auto-sampler and a column compartment (Infinity 1260, Agilent Technologies, Santa Clara, USA) coupled with a triple quadrupole mass detector (6410 QQQ, Agilent Technologies, Santa Clara, USA). The chromatography separation was performed on an XBridge BEH C18 column (particle size 2.5 μm, dimensions 4.6 × 150 mm) (Waters, Milford, Massachusetts, USA). The mobile phase consisted of 0.1%-acetic acid (constituent A) and acetonitrile (constituent B). The flow rate was 0.8 mL/min and the temperature was set at 40°C. A gradient elution program was employed: 0–3 min 90% A, 18 min 10% A, 18.1 min 90% A with a 3-minute post-run time. Mass spectrometry conditions were as follows: electrospray ionisation (ESI), positive polarity, source temperature 350°C, gas flow 9 L/min, nebulizer 45 psi, and capillary voltage 6 kV. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. **Table 1** shows the ions monitored within the frame of LC-MS/MS analyses targeted at T-2 and HT-2 toxin. The final concentrations of T-2 and HT-2 toxin were calculated based on the dilution

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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factor and the average recovery values obtained during the validation process.

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

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

**Mycotoxin Precursor ion Fragmentor voltage (V) Product ions Collision energy (eV) Cell accelerator** 

245.1a 27

285.1b 20

**voltage (V)**

1

1

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

T-2 toxin 489.2 200 387.1b 20

HT-2 toxin 447.2 100 345.1a 18

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

(either 500 μg/L or 1000 μg/L).

A more intense ion—used as a quantifier.

A less intense ion—used as a qualifier.

a

b

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 stored at 4°C pending analyses.
