4000 qtrap mass spectrometer

Cellular extracts were analyzed using the “AbsoluteIDQ p150 Kit” (Biocrates, Innsbruck, Austria). Briefly, samples were prepared as follows: (a) pipetting of 10 µl cell extract onto the filter inserts of the 96-well plate of the kit (containing internal standards labeled with stable isotopes), (b) drying of samples under a nitrogen stream, (c) extraction of metabolites and internal standards with 5 mM ammonium acetate in methanol, (d) centrifugation through the membrane filter provided, and (e) dilution with solvent for mass spectrometry. The final extracts were then randomized and analyzed using a 4000 QTrap mass spectrometer (Sciex) equipped with a TurboIonSpray source and coupled to an Agilent 1100 Series HPLC. Standard flow injection comprised two 20 µl injections (one for positive and one for negative electrospray ionization mode), and MRM was used for quantification as described earlier (27 (link)). Data were subsequently analyzed using the MetIQ software package (Biocrates, Innsbruck, Austria).

Analyses of bile acids was performed by Creative Proteomics Metabolomics (Shirley, NY) on flash frozen liver of liver-humanized mice. Method was provided by Creative Proteomics Metabolomics. An Agilent 1290 UHPLC system coupled to a Sciex 4000 Qtrap mass spectrometer was used. The MS instrument was operated in the multiple reaction monitoring mode with negative-ion detection. A Waters BEH C18 column (2.1 × 150 mm, 1.7 μm) was used and the mobile phase was (A) 0.01% formic acid in water and (B) 0.01% formic acid in acetonitrile for binary-solvent gradient elution. A mixture of standard substances containing all the targeted BAs was dissolved in 80% methanol to have a concentration of 20 μΜ for each bile acid. This solution was further diluted step by step at a same dilution ratio of 1 to 4 (v/v) with the same solvent to have 10-point calibration solutions. In all, 100 μL of each solution was then mixed with an equal volume of an internal standard (IS) solution containing 14 D-labeled bile acids. In all, 10 μL of each resultant solution was injected to run UPLC-ESI-MS/MS with the scheduled MRM. Linear regression calibration curves were constructed with the analyte-to-internal standard peak area ratios (As/Ai) versus molar concentrations (nmol/mL).

DON, ZEN and their respective metabolites were determined in heparinized plasma (S-Monovette®, Lithium-Heparin, Sarstedt AG & Co., Sarstedt, Germany) collected at day 71 (end of trial). Samples were measured with HPLC-MS/MS, using an Agilent 1200 series HPLC system (Agilent Technologies, Böblingen, Germany) coupled to a 4000 QTrap mass spectrometer (SCIEX, Foster City, CA, USA) described in Paulick et al. (2015b (link)). Analysis of DON, de-epoxy-DON (DOM-1), ZEN and its metabolites was performed according to Brezina et al. (2014 (link)) and of DONS1, 2 and 3 as described by Paulick et al. (2018 (link)). Limit of detection (LOD) and limit of quantification (LOQ) in plasma are provided in Table 1.