*2.5. Analysis of Trichothecenes*

Grain samples (60) were analyzed for the presence of trichothecenes according to Perkowski et al. [28]. Subsamples (10 g) were extracted with acetonitrile:water (82:18) and purified on a charcoal column (Celite 545/charcoal Draco G/60/activated alumina neutral 4:3:4 (*w*/*w*/*w*).

Type A trichothecenes (HT-2 toxin (HT-2), T-2 toxin (T-2), T-2 tetraol, T-2 triol, diacetoxyscirpenol (DAS), scirpentriol (STO)) were analyzed as TFAA (trifluoroacetic anhydride) derivatives. To the dried sample, 100 μL of trifluoroacetic acid anhydride were added. After 20 min, the reacting substance was evaporated to dryness under nitrogen. The residue was dissolved in 500 μL of isooctane and 1 μL was injected onto a gas chromatograph-mass spectrometer (GC/MS, Hewlett Packard GC 6890, Waldbronn, Germany). Type B trichothecenes (DON, NIV, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), fusarenon X (FUS-X)) were analyzed as TMS (trimethylsilylsilyl ethers) derivatives. To the dried extract, the amount of 100 μL of TMSI/TMCS (trimethylsilyl imidazole/trimethylchlorosilane; *v*/*v* 100/1) mixture was added. After 10 min 500 μL of isooctane were added and the reaction was quenched with 1 mL of water. The isooctane layer was used for the analysis and 1 μL of the sample was injected on a GC/MS system.

The analyses were run on a gas chromatograph (Hewlett Packard GC 6890, Waldbronn, Germany) hyphenated to a mass spectrometer (Hewlett Packard 5972 A, Waldbronn, Germany), using an HP-5MS, 0.25 mm × 30 m capillary column. The injection port temperature was 280 ◦C, the transfer line temperature was 280 ◦C and the analyses were performed with programmed temperature, separately for type A and type B trichothecenes. The type A trichothecenes were analyzed using the following programmed temperatures: Initial 80 ◦C held for 1 min, from 80 ◦C to 280 ◦C at 10 ◦C min−1, the final temperature being maintained for 4 min. For the type B trichothecenes initial temperature of 80 ◦C was held for 1 min, from 80 ◦C to 200 ◦C at 15 ◦C min−<sup>1</sup> held for 6 min and from 200 ◦C to 280 ◦C at 10 ◦C min−1, with the final temperature being maintained for 3 min. The helium flow rate was held constant at 0.7 mL min−1.

Quantitative analysis was performed in the single ion monitored mode (SIM) using the following ions for the detection of STO: 456 and 555; T-2 tetraol 455 and 568; T-2 triol 455 and 569 and 374; HT-2 455 and 327; T-2 327 and 401. DON: 103 and 512; 3-AcDON: 117 and 482; 15-AcDON: 193 and 482; NIV: 191 and 600. Qualitative analysis was performed in the SCAN mode (100–700 amu). Recovery rates for the analyzed toxins were as follows: STO 82 ± 5.3%; T-2 triol 79 ± 5.1%; T-2 86 ± 3.8%; T-2 tetraol 88 ± 4.0%; HT-2 91 ± 3.3%; DON 84 ± 3.8%; 3AcDON 78 ± 4.8%; 15 AcDON 74 ± 2.2%; and NIV 81 ± 3.8%. The limit of detection was 0.01 μg kg−1.
