6470 triple quadrupole mass spectrometer

For LC–MS/MS 1400W quantification, the liquid chromatography separations were performed with an Agilent Technologies 1290 Infinity II UHPLC instrument equipped with an Agilent ZORBAX RRHD HILIC Plus analytical column (2.1 mm × 100 mm, 1.8 µm) that was coupled to a 6470 triple quadrupole mass spectrometer with an atmospheric-pressure chemical ionization (APCI) source (Agilent Technologies, Santa Clara, CA). The chromatography was carried out at 40 °C with a flow rate of 0.400 mL/min. Data evaluation and peak quantitation were performed using Agilent MassHunter Qualitative Analysis (version 10.0) and Agilent MassHunter Quantitative Analysis (version 10.0) software (Agilent Technologies, Santa Clara, CA). Target peaks were found at 3.5 min retention time for 1400W and 3.25 min for the internal standard. 1400W quantification was finally determined by relative abundance to the internal standard and 1400W standard curve, before being made relative to the measured sample mass and volumes.

Polar metabolites were resuspended in 60% acetonitrile. Metabolites were measured using a Dionex UtiMate 3,000 LC System (Thermo Scientific) combined with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) and run in negative mode, using mass spectrometry based on accurate mass. Samples were injected onto a SeQuant ZIC/pHILIC Polymeric column (Merck Millipore)50 (link). The solvent, composed of acetonitrile and ammonium acetate (pH=9.3, 10 mM), was used at a flow rate of 0.100 ml min⁻¹. Data analysis was performed with the Xcalibur software. Metabolite levels were normalized to a fully ¹³C-labelled yeast extract and protein content. Alternatively, targeted measurements of polar metabolites were performed with a 1,290 Infinity II HPLC (Agilent) coupled to a 6,470 triple quadrupole mass spectrometer (Agilent). Samples were injected onto a iHILIC-Fusion(P) column with the above-mentioned solvents. Data analysis was performed with the Agilent Mass Hunter software.

All of the LC–MS analyses were performed using an analytical scale Agilent 1290 uHPLC system combined with an Agilent 6470 triple quadrupole mass spectrometer. The separations were performed at 35 °C on a Zorbax Eclipse Plus C18 column (50 × 2.1 mm, 1.8 μm particle size, 95 Å pore size) with a flow rate of 0.4 mL/min. The mobile phase consisted of H₂O (solvent A), and MeCN (solvent B), both acidified with 0.1% formic acid. The samples were separated using a 15-min program which was started at 2% B for 0.5 min, increased to 100% B for 9.0 min, kept at this level for the next 3.0 min, then reduced to 2% B for 1 min and finally re-equilibrated for 1.5 min. The injection volume was 2 μL. The mass spectrometer was equipped with an ESI source. The mass spectra were acquired in both positive and negative ionization modes, using a gas temperature of 250 °C, a gas flow of 5 L/min, a capillary voltage of 4000 V, a nebulizer pressure of 30 PSI, a sheath gas heater of 400 °C, a sheath gas flow of 12 L/min, and a nozzle voltage of 1000 V. Chromatographic separation and mass spectrometry were controlled using the Mass Hunter software (B.09.00, Agilent Technologies, Santa Clara, CA, USA).

An Agilent 1290 ultrahigh-performance liquid chromatography system coupled with a 6,470 triple–quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the complex mixture of phospholipids. A ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm; Agilent Technologies) column was used, maintaining the temperature at 50°C. The capillary voltage was set at 4.0 kV (positive ion mode) and 3.5 kV (negative ion mode). The sheath gas flow was set at 11 L/min.
All data were processed using Mass Hunter software (Agilent Technologies; B.08.00). The concentrations of phospholipid species were calculated from their relative abundances relative to the internal standard of each phospholipid class.