Using a repeated measures design, the rats (n = 6) received water, DON (2.0 mg/kg b.w.) and the equimolar amount (6.8 μmol/kg b.w.) of D3G (3.1 mg/kg b.w.) by gavage on days 1, 8 and 15 of the experiment, respectively. Stock solutions of 400 μg/mL DON and 619 μg/mL D3G were prepared by dissolving the solid standards in water. Thereof, volumes of 1.4–1.8 mL were administered to the rats according to

their weight. Feed was withdrawn 12 h before the treatment. After administration, the animals were housed individually in polycarbonate metabolic cages (Tecniplast, Hohenpeißenberg, Germany) for 48 h. Urine and feces were collected for the periods 0–24 h and 24–48 h after dosing and volumetrically measured or weighted, respectively. The samples were frozen at −20 °C at the check details end of the 48 h period. The study design was approved by both, the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Science and Research. Urine samples were centrifuged (10 min, 14,000 × g), acidified with 1% of acetic acid and cleaned up by solid phase extraction (SPE) on Strata C18-T cartridges (200 mg, Phenomenex, Aschaffenburg, Germany). After conditioning of the

cartridges with 5 mL of MeOH and 5 mL of MeOH/water/acetic acid (5/94/1, v/v/v), 500 μL of urine samples containing 1% of acetic acid were applied. Subsequently, the cartridges were washed with 1 mL of MeOH/water/acetic acid (5/94/1, v/v/v). The analytes were eluted with 2 mL of MeOH/water/acetic acid (70/29/1, v/v/v) selleck screening library and the eluates were evaporated to dryness under compressed air. The residues were reconstituted in 5 mL of ACN/water (20/80, v/v) for LC–MS/MS analysis. Feces samples were freeze-dried,

homogenized and 250 mg aliquots were extracted three times (40/40/20 min) with MeOH/water (50/50, Coproporphyrinogen III oxidase v/v, 3/2/2 mL) on a GFL rotary shaker (Burgwedel, Germany). 500 μL aliquots of the pooled raw extracts were combined with 500 μL cold MeOH. Subsequently, the solutions were vortexed for 15 s and centrifuged at 9000 × g for 10 min. Finally, 300 μL of the supernatants was evaporated to dryness under compressed air and re-dissolved in 300 μL of MeOH/water (20/80, v/v). The samples were vortexed for 30 s and clarified by centrifugation (10 min, 14,000 × g) for LC–MS/MS analysis. Clean-up procedures for feces and urine as described above resulted in sample dilutions by a factor of 56 and 10, respectively. Analysis was performed on an 1100 series high performance liquid chromatography (HPLC) system (Agilent Technologies, Waldbronn, Germany) coupled to a QTrap 4000 LC–MS/MS System (AB Sciex, Foster City, CA) equipped with a Turbo V electrospray ionization (ESI) source. Chromatographic separation was achieved on an Atlantis® T3 column (3.0 mm × 150 mm, 3 μm, Waters, Vienna, Austria) equipped with a 4 mm × 3 mm C18 security guard cartridge (Phenomenex, Torrance, CA, USA). Eluent A was composed of water/acetic acid (99.9/0.