5890 gas chromatograph

1D- and 2D-NMR spectra were recorded in CDCl₃ with a Bruker AVANCE II 400 spectrometer (Bruker, Billerica, MA, USA) operated at 400 MHz for ¹H and at 100 MHz for the ¹³C nucleus. Tetramethylsilane (TMS) was used as the internal reference (δ 0.00). The high resolution mass spectra were determined in a Bruker MicroQTOF-II mass spectrometer (Bruker), equipped with an ESI source operated in positive mode at 180 °C with a capillary voltage of 4500 V. Mass accuracy was verified by calibration before and after sample introduction, using sodium formate (1 mM). Both samples and calibrant were introduced using a syringe pump at 10 µL min⁻¹. Electron impact mass spectra (EI-MS) were obtained at 70 eV by GC–MS on a Hewlett–Packard 5970 Series mass spectrometer interfaced (Hewlwtt-Packard, Palo Alto, CA, USA) with a Hewlett–Packard 5890 gas chromatograph fitted with a column (HP-5MS, 15 m × 0.25 mm i.d., temperature from 200–290 °C, 10 °C/min). HPLC was performed in a Shimadzu chromatograph with a diode array UV-Vis detector (Shimadzu, Kyoto, Japan). Fourier transform infrared spectra were acquired on a FTIR Nicolet 510P spectrometer (Thermo Scientific, Waltham, MA, USA). Spectra over a range of 4000–500 cm⁻¹ with a resolution of 2 cm⁻¹ (50 scans) were recorded using KBr pellets.

The quantitative analysis of the total AFs (B1, B2, G1, and G2) in corn samples and corn samples after bioethanol production (the stillage obtained after drying at 50°C for 24 h) was performed by a competitive enzyme linked immunosorbent assay (ELISA) according to the total AF test (AgraQuant^®, Romer Labs Ltd., Germany) procedure. The ground test sample amount used in the ELISA assay was 100 g. Mycotoxin extraction and testing was carried out according to the manufacturer’s instructions.
Acidity analysis of fermented broth in bioethanol production was performed according to our previous study (Juodeikiene et al., 2012 (link)). The concentration of ethanol was determined using direct distillation and pycnometry.
Volatile compound determination was completed by gas chromatography (GC). Corn samples with different contamination levels were used for bioethanol production (Table 1). The bioethanol production was performed by using the low-temperature process according to Juodeikiene et al. (2014a) (link). A Hewlett-Packard 5890 gas chromatograph equipped with an FID detector was used for the quantitative analysis of volatile compounds as described by Juodeikiene et al. (2014a) (link).

Lipids were extracted following the technique proposed by Folch et al. [44 (link)]. The conversion of lipids into FA methyl esters (FAMEs) was performed by acid-catalysed transesterification of total lipids according to the method of Christie [45 ]. The total lipid samples were transmethylated overnight in 2 mL of 2% sulphuric acid in methanol (plus 1 mL of toluene to dissolve neutral lipids) at 50 °C. Methyl esters were extracted twice in 5 mL of hexane–diethyl ether (1:1, v/v) after neutralisation with 2 mL of 2% KHCO₃, dried under nitrogen and redissolved in 1 mL of iso-hexane. FAMEs were separated and quantified by gas-liquid chromatography in an SP™ 2560 flexible fused silica capillary column (length 100 m, internal diameter 0.25 mm, film thickness 0.20 mm SUPELCO) in a Hewlett–Packard 5890 gas chromatograph. The 140 °C oven temperature was initially increased at a rate of 3 °C min⁻¹ to 230 °C, followed by 2 °C min⁻¹, and then to 240 °C to be held for 12 min. The injector and flame ionisation detector were set at 260 °C. Helium was used as the carrier gas at a pressure of 300 kPa. Peaks were identified by comparing their retention times to appropriate FAME standards from the Sigma Chemical Company (St. Louis, MO, USA). Each component’s data were reported as a percentage of total content [8 (link),46 (link)].