Multiplex Analysis of Mycotoxins in Food Samples

The analyses were carried out using methods developed in our department; in particular, AFs were determined by HPLC-FLD as reported by Bertuzzi et al. [36 (link)]; OTA by HPLC-FLD [37 (link)], DON by GC-MS [38 ], STC by LC-MS/MS [13 (link)]. Briefly, AFs were extracted from 25 g of sample with 250 mL of acetone-water 7 + 3 v/v using a rotary-shaking stirrer for 60 min. After purification through an immunoaffinity column, the extract was filtered (Millex HV 0.45 mm) before HPLC-FLD analysis (Jasco Corporation, Tokyo, Japan); the average recovery for AFB₁ was 95.8 ± 3.4% (three replicates at two spiking levels, 2.0 and 10.0 µg kg⁻¹). The limit of detection (LOD) and quantification (LOQ), defined at those levels resulting in signal-to-noise ratios of 3 and 10, were 0.05 and 0.15 µg kg⁻¹, respectively. OTA was extracted from a 10 g of sample with 100 mL of a mixture of 0.13 M sodium bicarbonate-methanol (5 + 5 v/v) for 45 min using a rotary-shaking stirrer. After purification through an immunoaffinity column, the eluate was concentrated under a gentle stream of nitrogen, brought to 2 mL with acetonitrile-water (41 + 59 v/v), vortex-mixed for few seconds and filtered before HPLC-FLD analysis. The mean recovery (mean of 3 replicates at 2 spiking levels) was 95.2 ± 3.4%; LOD and LOQ were 0.02 and 0.06 µg kg⁻¹, respectively. DON was extracted from samples (25 g) with 100 mL of acetonitrile-water (86 + 14 v/v); an aliquot (6 mL) of the filtrate was slowly pressed through a MycoSep 227 column. An aliquot (200 µL) of the internal standard diacetoxyscirpenol (DAS 10 mg L⁻¹) was added to 2 mL of the eluate. The solution was evaporated to dryness and derivatized with 200 µL of trimethilsilylimidazole-trimethilclorosilane (1 + 0.2 v/v) for 15 min in subdued light. Then 0.8 mL hexane was added, and the solution was washed with 1 mL 0.2 M phosphate buffer pH 7.5, and the hexane phase was used for GC-MS. GC-MS analysis was carried out using a TraceGQ Ultra coupled with an ISQ single quadrupole mass spectrometry (Thermo-Fisher Scientific, San Jose, CA, USA). The analysis was carried out using a capillary column Rtx-5MS, 30 m × 0.25 mm i.d., 0.25 µm film thickness. LOD and LOQ were 5 and 15 µg kg⁻¹, respectively; the average recovery was 92.4 ± 2.6%. STC was extracted from an aliquot of 20 g taken from the milled sample with 100 mL acetonitrile-water 8 + 2 v/v using a rotary-shaking stirrer for 60 min. After purification through an immunoaffinity column, the extract was concentrated under a gentle flow of nitrogen and brought to 1 mL with acetonitrile-water 4 + 6 v/v. An aliquot of 900 µL of cleaned extract was transferred into an autosampler vial and mixed with 100 µL of isotopically labelled STC (12 µL L⁻¹). A volume of 20 µL of the extract was injected into an LC-MS/MS system consisting of a LC 1.4 Surveyor pump, a Quantum Discovery Max triple-quadrupole mass spectrometer (Thermo-Fisher Scientific, San Jose, CA, USA) and a PAL 1.3.1 sampling system (CTC Analytics AG, Zwingen, Switzerland). STC was chromatographed on a Betasil RP-18 column (5 µm particle size, 150 × 2.1 mm, Thermo-Fisher) with a gradient acetonitrile-water (both acidified with 0.2% formic acid; flow rate 0.2 mL min⁻¹); the ionization was performed using positive atmospheric pressure chemical ionization (APCI). The matrix effect was low, less than 10%; the LOD and the LOQ were 0.05 and 0.15 µg kg⁻¹, respectively. The average recovery was 90.4% ± 4.2%.