Esquire 6000 esi ion trap mass spectrometer

The O-methylflavonoid content of E. coli culture extracts was analyzed using an Agilent 1100 Series LC system (Agilent Technologies) coupled to an ultraviolet diode array detector (UV-DAD, Agilent Technologies) and an Esquire 6000 ESI-Ion trap mass spectrometer (Bruker Daltonics). Chromatographic separation was performed on an EC 250/4.6 Nucleodur Sphinx column (RP 5 μm, Macherey-Nagel, Düren, Germany), with 0.2% (v/v) formic acid in water (A) and acetonitrile (B) as mobile phases. The flow rate was 1 mL/min and the column temperature was set to 25°C. The following elution profile was used: 0–15 min, 30–60% B; 15.1–16 min, 100% B; 16.1–20 min, 30% B. The mass spectrometer was run in alternating ion polarity (positive/negative) mode with a skimmer voltage of +40 V/−40 V, a capillary voltage of −3,500 V/+3,000 V and a capillary exit voltage of 113.5 V/−113.5 V, to scan masses from m/z 50–3,000. N₂ was used as drying gas (11 L/min, 330°C) and nebulizer gas (35 psi). The software programs esquireControl version 6.1 (Bruker Daltonics) and HyStar version 3.2 (Bruker Daltonics) were used for data acquisition, while DataAnalysis version 3.4 was used for data processing. The UV absorption of individual O-methylflavonoids was analyzed using the post-processing software included in the HyStar version 3.2 package (Bruker Daltonics).

Comparative HPLC-MS (ion trap) analyses of aqueous feces extracts were carried out on an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) coupled to an Esquire 6000 ESI-Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in alternating ionization mode in the range m/z 55–1000. Skimmer voltage −40 eV, capillary exit voltage −102.3 eV, capillary voltage 4,000 V, nebulizer pressure 35 psi, drying gas, 11 l/min; gas temperature 330°C. MS² and MS³ spectra of candidate metabolites were obtained using the AutoMS mode of the software (Bruker Daltonics). Separation was accomplished using a Nucleodur Sphinx RP column (250 × 4.6 mm, 5 μm particle size, Macherey-Nagel) with a mobile phase consisting of 0.2% (v/v) formic acid in ultrapure water as solvent A and acetonitrile as solvent B with a flow rate of 1.0 ml/min at 25°C. The gradient was as follows: 0–100% (v/v) B (25 min), 100% B (3 min), 100–0% (v/v) B (0.1 min), 0% B (4.9 min). Eluent flow rate was diverted in a ratio of 4:1 before entering the ESI unit. Chromatograms were analyzed with the DataAnalysis and MetaboliteTools software packages from Bruker Daltonics. Candidate metabolites were further analyzed using MS/MS and high resolution mass spectrometry (see section UHPLC-ESI-MS/MS).