Fig 5.

(A) DON concentrations in plasma over time – Mice were administered DON at 1.6 mg/kg (i.v.). Mice were euthanized and blood was collected transcardially 0.25, 0.5, 1, 2, 4 and 6 hours after dosing. Blood was centrifuged, plasma collected and stored at −80 °C. N-butanol containing 3N HCl (250 μL) was added directly to samples (50 μL) and centrifuged at 16,000 × g for 5 minutes to precipitate proteins. An aliquot (200 μL) of the supernatant was incubated at 60 °C for 30 min. After derivatization, the samples were analyzed by LC-MS (methods). (B) DON Plasma to Brain ratio analysis – Mice were administered DON (0.6 mg/kg, i.p.) and blood and brain were collected 1 h after DON administration. Plasma was isolated from blood samples as described in (A). Brains were collected following perfusion with PBS and frozen immediately at −80 °C. Before bioanalysis, brains were weighed and n-butanol containing 3N HCl was added to each sample (5 μL/mg tissue) and homogenized using a pestle. Resulting homogenates were centrifuged at 16,000 × g for 5 minutes to precipitate proteins and analyzed the same way as the plasma samples. All determinations in both (A) and (B) were carried out in triplicate. Error bars correspond to ± S.E.M.