Methanol

The sample powder was mixed with methanol (JT Baker Center Valley PA USA) solution (CH₃OH 80%), then homogenized (mixing shaker IKA Werke Staufen Germany) and centrifuged (J-HC centrifuge Beckman Coulter USA). The supernatant was dissolved in PBS (all reagents were from Sigma Aldrich Saint Luis MO USA) and cleaned up by an IAC (Aflastar, ROMER Labs DH Erber Campus Getzersdorf Austria). The IAC was eluted with 2 mL of methanol acetic acid solution (JT Baker Center Valley PA USA) (CH₃OH: 2% CH₃COOH). The total amount of 2 mL was analysed in LC-MS/MS (Liquid chromatography–mass spectrometry) (Waters corporation, Milford, MA, USA) with a binary gradient of water and acetonitrile with 0.2% formic acid in a reverse-phase high Performance LC column (Luna C18, 5 µ, 2.1 mm × 150 mm; Phenomenex inc., Torrance, CA, USA). Fragmentation was performed in positive ionization. The quantification was on mass/charge 241.0 for AFB₁, 243.0 for AFB₂, 200.0 for AFG₁ and 188.9 for AFG₂. The sensitivity of the method was 0.5 µg/kg for each aflatoxin. The analytical method was previously validated for food matrices and applied to dust samples.

This determination made it possible to observe probable changes in the fatty acid composition due to the MDDM obtention process [39 (link)]. Firstly, 10 g of raw material or 2 g of MDDM was homogenized with an Ultraturrax device (T-25 basic, IKA-WERKE) using different solvents: chloroform (Carlo Erba), methanol (Carlo Erba), potassium chloride (Panreac), and water. This mixture was centrifuged for 10 min at 4000 rpm (Mod. Universal 320 R, Hettich, Tuttlingen, Germany), and the fat was extracted from the top. After incorporating BHT (Sigma-Aldrich) as antioxidant, solvents were evaporated with nitrogen gas. Afterwards, methylation was performed using 0.03 g of this previous fat. This fat was mixed with an intern pattern, C23:0 (TCI), which does not caused interferences in the matrix. Then, 2 mL of hexane (Carlo Erba) and 1 mL of potassium hydroxide (Panreac) saturated solution in methanol was added. The upper phase was used. To analyze the fatty acid profile, a gas chromatograph (HP-6890 II, Hewlett-Packard, Palo Alto, CA, USA) was employed with a column SP-2380 (100 m × 0.25 mm × 0.20 µm). The temperature program was 140–165 °C at 3 °C/min during 10 min and 165–220 °C at 5 °C/min during 50 min. Fatty acid content was quantified as total area (%) of identified fatty acids.