Kinetex pfp column

Tocopherols were quantified in plasma (collected at slaughter), liver, and endometrium by a validated method described earlier [62] . Briefly, tissue samples were saponified for 30 min at 70 • C in a shaking water bath. To both tissue and plasma samples butylated hydroxytoluene (BHT; 25 μL of a 1 mg/ml ethanolic solution) was added. Then all samples were extracted twice with n-Hexane. The supernatants were pooled and evaporated (Christ SpeedDry; Christ, Osterode Germany). The dried residues were resuspended and injected into a Jasco HPLC system (AS-950 Plus autosampler, PU-980 Plus pump, FP-950 Plus fluorescence detector, LG-980-02 gradient unit, and a 3-line degasser; Jasco, Groß-Umstadt, Germany). The tocopherols were separated on a Kinetex PFP column (2.6 μm, 150 × 4.6 mm; Phenomenex, Aschaffenburg, Germany) using a methanol: water (85:15, vol/vol) mobile phase. The fluorescence detector was set to an excitation wavelength of 296 nm and emission wavelength of 325 nm. Peaks were recorded and integrated using ChromPass version 1.8.6.1 (Jasco). The concentrations of tocopherols were quantified against external standard curves with authentic compounds (Sigma Aldrich, St. Louis, MO).

Samples taken from the enzyme reactions for HPLC and LC-MS analysis were thoroughly mixed with dichloromethane in order to stop the reactions. The water phase obtained after centrifugation (16 000 3 g, 5 min) was used for analysis. For routine analysis of 2-hydroxyisobutyryl-CoA and 3-hydroxybutyryl-CoA, a Kinetex PFP column (5 mm, 100 A ˚, 250 3 4.6 mm; Phenomenex, USA) was used on a Shimadzu Prominence system with PDA detector (SPD-M20A). The eluents were 100 mM ammonium acetate (eluent B) and acetonitrile (eluent A). The method started with 5% eluent A for 5 min, followed by a gradient from 5% to 25% eluent A between 5 and 19 min; the flow rate was 0.75 ml min 21 and the column was maintained at a temperature of 408C. The injection volume was 5 ml.

Tocopherol extractions and analyses were performed following the procedures described in Bruni et al. (2001) (link). In particular, the same amaranth flour amount for each sample (5 g), reported in the "Field Experiments and Plant Material" section, was placed in a flask containing 100 mL of methanol and stirred for 24 h in the dark at a constant temperature of 25°C. The samples were then filtered and centrifuged for 20 min (3000 rpm). The supernatant was recovered, dried with rotavapor, and weighed. Tocopherol analyses of the extracts were performed employing a modular JASCO high performance liquid chromatography (HPLC) unit characterized by a PU-2089Plus pump, a MD-2010Plus diode array detector (DAD), and a 20-μL sampler loop. Isocratic elution mode (methanol:water; 85:15) was used with a Phenomenex KINETEX PFP column ( 15´ 0.46 cm, 2.6 μm). Flow rate was 0.8 mL min -1 while absorbance was detected at 295 nm. All solvents used were chromatographic grade. Vitamin E stereoisomer peaks (a-, b-, g-, d-tocopherols) were identified by comparing their retention time with those of pure standards (Sigma-Aldrich). Integration of the peak areas was performed using a dedicated Borwin software (Borwin-PDA ver. 1.50, JMBS Developments). For each amaranth extract, qualitative and quantitative analyses were performed in triplicate.

Mycotoxin extraction was developed as detailed in Estiarte et al. (2016) . Separation, detection and quantification of AOH and AME were performed on a HPLC system model 2510 HPLC pump (Varian, Inc., Palo Alto, CA) connected to one in-line Spectroflow 757 UV/Vis absorbance detector (Applied Biosystems, Foster City, CA). A reverse phase Kinetex PFP column (5 µm, 4.6 × 150 mm, Phenomenex, Torrance, CA, USA) preceded by a KrudKatcher classic HPLC in-line filter (0.5 µm depth filter, Phenomenex, CA, USA) were used. Chromatographic and method performance characteristics for AOH and AME detection and quantification are detailed in Estiarte et al. (2016) .