Ultrafree mc centrifugal device

One milliliter of MMO medium was weighed in a glass tube (10 mL). A calibration curve was set up by spiking three blank medium samples with DON, NIV, 3-ADON, 15-ADON, DOM-1 and DON-3G (10 ng/µL, range 100 µg/L–200 µg/L–400 µg/L). The stock solution (25 µL) of the internal standard ZAN (10 ng/µL, 250 ng on sample) was added to both calibrators as unknowns. Samples were vortexed (Labinco BV, Breda, The Netherlands) for 2 min. The medium samples were evaporated until dryness under nitrogen at 60 °C using the TurboVap^® LV (Biotage, Dusseldorf, Germany), and redissolved in 100 µL of injection solvent (methanol/water/acetic acid (41.8/57.2/1, v/v/v) with 5 mM of ammonium acetate). Finally, the redissolved sample was vortexed for 3 min. Prior to LC-MS/MS analysis, the samples were collected in an Ultrafree-MC centrifugal device (0.22 µm, Millipore, Bedford, MA, USA) and centrifuged for 10 min at 10,000 g.

In order to objectively indicate the validity of the obtained results, 20 different EDTA-anticoagulated blood samples (5 mL) were collected from Rode Kruis (Ghent, Belgium). The use of the human samples was approved by the Ethics Committee of Ghent University Hospital (B670201630414). Each sample was analyzed following 2 different protocols: (1) the VAMS methodology as described in this manuscript and (2) a liquid/liquid multi-mycotoxin extraction methodology, applied on liquid whole blood [44 (link)]. In brief, to 100 µL of blood, previously spiked with IS, 100 µL of acetonitrile was added in a 2 mL Eppendorf tube. Then, samples were vortexed for 20 s and shaken using an overhead shaker (Agitelec, J. Toulemonde and Cie, Paris, France) for 30 min. The Eppendorfs were then centrifuged at 9000× g for 6 min. Next, 160 µL of the supernatant was evaporated to dryness (N2, 40 °C). Finally, the dry residue was redissolved in 100 µL of the injection solvent (methanol/water, 60/40, v/v), vigorously vortexed and subjected to centrifugation (Ultrafree^®-MC centrifugal device, Millipore, Bedford, MA, USA) for 10 min at 5000 g. Finally, samples were transferred into vials for analysis and 5 µL were injected into the LC-MS/MS.

A 4.00 ± 0.02 g homogenized sample was weighed in a 50 mL extraction tube. The samples were spiked to construct a matrix-matched calibration curve at the following CIT/HO-CIT concentrations: 0.1–0.5–1–5–10–50–100–200 µg/kg. To each sample, ¹³C₁₃-CIT was added at a concentration of 5 µg/kg After leaving the samples 30 min for equilibration, 10 mL of an acidified saline solution was added, consisting of 10% (m/v) NaCl in acidified water (1.6% HCl in H₂O/HAc (99/1, v/v)), followed by 20 mL of extraction mixture consisting of EtAc, ACN and HAc (75/24/1, v/v/v). Samples were extracted for 1 h at room temperature using an overhead shaker (Agilitec, J. Toulemonde and Cie, Paris, France). Subsequently, 6.0 ± 0.2 g of MgSO₄ and 1.5 ± 0.1 g of NaCl were added. Samples were first agitated by hand for 30 s to avoid aggregation of the salts, followed by shaking using the overhead shaker for 3 min. Next, samples were centrifuged for 5 min at 3676× g. An aliquot of 1 mL supernatant was transferred and evaporated to dryness under a gentle stream of nitrogen at 40 °C. Extracts were reconstituted in 0.2 mL of injection solvent (MeOH, water 50/50 v/v), vigorously vortexed for 1 min and subjected to ultracentrifugation (Ultrafree^®-MC centrifugal device; Millipore, Bedford, MA, USA) for 10 min at 9167× g. Finally, extracts were transferred into autosampler vials for analysis.